Cell-based method for determining an activity of botulinum toxin

ABSTRACT

A new cell line and an antibody for determining the activity of botulinum toxin are disclosed. Also disclosed is a method of determining the activity of botulinum toxin using the cell line and/or the antibody.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 17/029,892filed Sep. 23, 2020, which is a divisional of application Ser. No.16/245,415 filed Jan. 11, 2019, now U.S. Pat. No. 10,823,725 issued Nov.3, 2020, which claims priority based on Korean Patent Application No.10-2018-0150640 filed Nov. 29, 2018, Korean Patent Application No.10-2018-0150997 filed Nov. 29, 2018; and Korean Patent Application No.10-2018-0159701 filed Dec. 12, 2018, the contents of which areincorporated herein by reference in their entireties.

SEQUENCE LISTING

The content of the electronically submitted sequence listing, file name:Sequence_Listing_As_Filed.txt; size: 40,208 bytes; and date of creation:Sep. 21, 2021, filed herewith, is incorporated herein by reference inits entirety.

TECHNICAL FIELD

The present invention relates to a cell-based method for determining anactivity of botulinum toxin.

BACKGROUND ART

At present the mouse LD₅₀ bioassay (mLD₅₀) is a generally acceptedmethod for detection of BoNT/A remaining in food, clinical orenvironmental samples. Especially, pharmaceutical industry uses mLD₅₀ asa standard assay to measure BoNT/A potency for aesthetic or clinicalapplications. However, the BoNT/A potency estimated by mLD50 are knownto vary to a great extent depending on testing institutes/facilities andresearchers, it is challenging to accurately and reproducibly quantifythe biological potency of BoNT/A.

ZEBET meeting was held on April 27-28, in Berlin, Germany, to promotealternative approaches to mLD₅₀ in order to standardize the measurementof BoNT/A and also to minimize the number of test animals involved andthe pain caused to them (Altern Lab Anim. 2010 August; 38(4):315-30).Consequently, many research institutes and industries in the world haveembarked diverse researches to develop cell-based potency assays (CBPAs)or cell-based bioassay (CBB) which could substitute for mLD₅₀ withsatisfactory specificity, sensitivity and reproducibility. In order tosuccessfully establish CBPA or CBB, they have been attempting to acquire(1) monoclonal or polyclonal antibodies specific for SNAP25₁₉₇ and (2)neuronal cell lines that exhibit high sensitivities to low levels ofBoNT/A (˜pM).

As early as in 2004, Dr. Chapman and his coworkers invented afluorescent reporter assay that utilized epigenetically expressed SNAP25fused to two fluorescent proteins (Proc Natl Acad Sci USA. 2004 Oct. 12;101(41):14701-6), which is the technology platform of BoCell™(BioSentinel Inc). Although it is the first of its kind that enables thedetection of BoNT/A endopeptidase activity in mammalian cells growing inthe 96-well culture plate, BoCell™ assay is about 2-3 orders ofmagnitude less sensitive than mouse bioassay (Appl Environ Microbiolvol. 78, 21 (2012): 7687-97).

As briefed above, there have been worldwide efforts to develop CBPAs toreplace mLD50, ultimately eliminating the animal testing. Althoughseveral institutes have developed CPBA, there remain few drawbacks toovercome. Invention of much improved CBPA will not only facilitatedevelopment of diverse BoNT/A-related products but also strengthen thecompetitiveness of the products by giving higher levels of confidence toconsumers with regards to the quality control. On the basis of thispremise, we have taken multi-facet approaches to develop a more reliableCBPA.

Therefore, the present invention is directed to a cell, antibodies, anda method for determining an activity of botulinum toxin. Firstly, weemployed a very stringent strategy to produce monoclonal antibodies withmuch higher binding affinity and specificity toward SNAP25. Of severalantibodies obtained using synthetic peptides, B4 IgG and C16 IgG wereselected as capture and detection antibody for CBPA, respectively. B4IgG has a high affinity and equal specificity toward full-length SNAP25and its cleaved form, SNAP25₁₉₇, generated by BoNT/A, whereas C16 IgG ishighly specific for SNAP25₁₉₇ with no significant binding toSNAP25_(FL). Secondly, through comparative analysis of 13 differentneuronal cell lines, followed by an extensive clonal selection, a novelcell line, N2-42F, was established. In addition to BoNT/A sensitivitythat can be compared to that of SiMa, ˜24 hr of PDT and stableattachment to poly-d-lysine (PDL)-coated culture plate make N2-42F cellsa very attractive and reliable host for CBPA. Thirdly, CBPA wasoptimized using N2-42F cells, B4 IgG as capture antibody, and C16 IgG asdetection antibody, with which as low as 0.5 U potency of BoNT/A wasmeasured per assay. Passage stability and stable maintenance/storage ofN2-42F cells, and exclusive use of monoclonal antibodies as both captureand detection antibodies make this novel CBPA very reliable andreproducible.

DISCLOSURE Technical Problem

The present invention has been made in order to solve theabove-described problems occurring in the prior art, and it is an objectof the present invention to provide a cell, antibodies, and a method fordetermining an activity of botulinum toxin.

However, the technical object to be achieved by the present invention isnot limited to the above-mentioned technical object, and other objectsthat are not mentioned above can be clearly understood by those skilledin the art from the following description.

Technical Solution

Hereinafter, various embodiments described herein will be described withreference to figures. In the following description, numerous specificdetails are set forth, such as specific configurations, compositions,and processes, etc., in order to provide a thorough understanding of thepresent invention. However, certain embodiments may be practiced withoutone or more of these specific details, or in combination with otherknown methods and configurations. In other instances, known processesand preparation techniques have not been described in particular detailin order to not unnecessarily obscure the present invention. Referencethroughout this specification to “one embodiment” or “an embodiment”means that a particular feature, configuration, composition, orcharacteristic described in connection with the embodiment is includedin at least one embodiment of the present invention. Thus, theappearances of the phrase “in one embodiment” or “an embodiment” invarious places throughout this specification are not necessarilyreferring to the same embodiment of the present invention. Additionally,the particular features, configurations, compositions, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless otherwise specified in the specification, all the scientific andtechnical terms used in the specification have the same meanings ascommonly understood by those skilled in the technical field to which thepresent invention pertains.

Allergan Inc., the manufacturer of Botox®, successfully producedmonoclonal antibodies specific for SNAP25₁₉₇ and also identified a humanneuroblastoma SiMa as an ideal host cell line highly sensitive to BoNT/A(PLoS One. 2012; 7(11):e49516). Using these reagents, they developed anovel CBPA to assess the stability and potency of BoNT/A, which wasapproved by FDA to replace mLD50 for the first time in 2010 (US patentsU.S. Pat. No. 8,455,213B2 and US2010/0204559A1). MERZ Pharma, the Germanmanufacturer of Botulinum Toxin, developed CBA-ELISA in 2014 (WO2014/207109A1) and obtained FDA approval in 2015. CBA-ELISA employsin-situ fixation of differentiated neuronal cells, followed by membranepermeablization and immunological detection of endogenous SNAP25(Synaptosomal nerve-associated protein 25).

Allergan's CBPA and MERZ's CBA-ELISA exhibit excellent sensitivitieswith sub-picomolar concentrations of EC₅₀ (i.e. <1.0 U/well) that areequivalent to mouse bioassay. Allergan's and MERZ's technology platformcommonly utilize a commercial rabbit polyclonal antibody (Sigma S9684)to detect SNAP25 under their optimal conditions. Differentiated humanneuroblastoma SiMa cells are exclusively used in Allergan's CBPA, whilehuman differentiated induced pluripotent stem cells (iPS) are used ashost in the standardized and optimized MERZ's CBA-ELISA. SiMa growsslowly with over 70 hrs of population doubling time (PDT). Similarly,the generation of human neuronal differentiated iPS cells istime-consuming, and moreover their storage is as difficult asgeneration. Thus, CBPA would become more reliable if a neuronal cellline is not only highly sensitive to BoNT/A but can be easily maintainedand stored with a faster PDT. Moreover, a research group led by Dr.David Beebe at the University of Wisconsin has cast a question on thesuitability of SiMa cell since it does not exhibit motor neuron-likecharacteristics (J Biomol Screen. 2016 January; 21(1):65-73). Theydeveloped an alternative CBPA using a motor neuron-like cell lineNG108-15 that exhibited EC₅₀ of ˜7.9 pM. Since CBPA relies on theWestern blot analysis to comparatively determine endogenous levels ofSNAP25₁₉₇ and SNAP25_(FL), its utilization as a high-throughput assayappears to be challenging at present.

In one embodiment of the present invention, “botulinum toxin” is aneurotoxic protein produced by the bacterium Clostridium botulinum. Thegenus Clostridium has more than 127 species, grouped according to theirmorphology and functions. The anaerobic, gram-positive bacteriaClostridium botulinum produces a potent polypeptide neurotoxin,botulinum toxin, which causes a neuroparalytic illness in humans andanimals referred to as botulism. The spores of Clostridium botulinum arefound in soil and can grow in improperly sterilized and sealed foodcontainers of home based canneries, which are the cause of many of thecases of botulism. The symptoms of botulism typically appear 18 to 36hours after eating the foodstuffs infected with a Clostridium botulinumculture or spores. The botulinum toxin can apparently pass unattenuatedthrough the lining of the gut and shows a high affinity for cholinergicmotor neurons. Symptoms of botulinum toxin intoxication can progressfrom difficulty in walking, swallowing, and speaking to paralysis of therespiratory muscles and death.

Botulinum toxin type A is known as the most lethal natural biologicalagent to man. About 50 picograms of a commercially available botulinumtoxin type A (purified neurotoxin complex) is an LD50 (i.e., 1 unit).Interestingly, on a molar basis, botulinum toxin type A is about 1.8billion times more lethal than diphtheria, about 600 million times morelethal than sodium cyanide, about 30 million times more lethal thancobra toxin and about 12 million times more lethal than cholera. Oneunit (U) of botulinum toxin is defined as the LD50 upon intraperitonealinjection into female Swiss Webster mice weighing 18 to 20 grams each.

Immunologically distinct 7 botulinum neurotoxins have been generallycharacterized as botulinum neurotoxin serotypes A, B, C1, D, E, F and G,each of which is distinguished by neutralization with type-specificantibodies. The different serotypes of botulinum toxin vary in theanimal species that they affect and in the severity and duration of theparalysis they evoke. For example, it has been determined that botulinumtoxin type A is 500 times more potent, as measured by the rate ofparalysis produced in the rat, than botulinum toxin type B.Additionally, botulinum toxin type B has been determined to be non-toxicin primates at a dose of 480 U/kg which is about 12 times the primateLD50 for botulinum toxin type A. Botulinum toxin apparently binds withhigh affinity to cholinergic motor neurons, is translocated into theneuron and blocks the release of acetylcholine. Additional uptake cantake place through low affinity receptors, as well as by phagocytosisand pinocytosis.

Regardless of serotype, the molecular mechanism of toxin intoxicationappears to be similar and to involve at least 3 steps. In the first stepof the process, the toxin binds to the presynaptic membrane of thetarget neuron through a specific interaction between the heavy chain(the H chain or HC), and a cell surface receptor. The receptor isthought to be different for each type of botulinum toxin and for tetanustoxin. The carboxyl end segment of the HC appears to be important fortargeting of the botulinum toxin to the cell surface.

In the second step, the botulinum toxin crosses the plasma membrane ofthe target cell. The botulinum toxin is first engulfed by the cellthrough receptor-mediated endocytosis, and an endosome containing thebotulinum toxin is formed. The toxin then escapes the endosome into thecytoplasm of the cell. This step is thought to be mediated by the aminoend segment of the heavy chain, the HN, which triggers a conformationalchange of the toxin in response to a pH of about 5.5 or lower. Endosomesare known to possess a proton pump which decreases intra-endosomal pH.The conformational shift exposes hydrophobic residues in the toxin,which permits the botulinum toxin to embed itself in the endosomalmembrane. The botulinum toxin (or at least the light chain of thebotulinum toxin) then translocates through the endosomal membrane intothe cytoplasm.

The last step of the mechanism of botulinum toxin activity appears toinvolve reduction of the disulfide bond joining the heavy chain and thelight chain. The entire toxic activity of botulinum and tetanus toxinsis contained in the light chain of the holotoxin; the light chain is azinc (Zn⁺⁺) endopeptidase which selectively cleaves proteins essentialfor recognition and docking of neurotransmitter-containing vesicles withthe cytoplasmic surface of the plasma membrane, and fusion of thevesicles with the plasma membrane. Tetanus neurotoxin, botulinum toxintypes B, D, F, and G cause degradation of synaptobrevin (also calledvesicle-associated membrane protein (VAMP)), a synaptosomal membraneprotein. Most of the VAMP present at the cytoplasmic surface of thesynaptic vesicle is removed as a result of any one of these cleavageevents. Serotype A and E cleave SNAP-25. Serotype C1 was originallythought to cleave syntaxin, but was found to cleave syntaxin andSNAP-25. Each of the botulinum toxins specifically cleaves a differentbond, except type B (and tetanus toxin) which cleave the same bond. Eachof these cleavages blocks the process of vesicle-membrane docking,thereby preventing exocytosis of vesicle content.

Botulinum toxins have been used in clinical settings for the treatmentof neuromuscular disorders characterized by hyperactive skeletal muscles(i.e. motor disorders). In 1989, a botulinum toxin type A complex wasapproved by the U.S. Food and Drug Administration for the treatment ofblepharospasm, strabismus and hemifacial spasm. Subsequently, abotulinum toxin type A was also approved by the FDA for the treatment ofcervical dystonia and for the treatment of glabellar lines, and abotulinum toxin type B was approved for the treatment of cervicaldystonia. Non-type A botulinum toxin serotypes apparently have a lowerpotency and/or a shorter duration of activity as compared to botulinumtoxin type A. Clinical effects of peripheral intramuscular botulinumtoxin type A are usually seen within one week of injection. The typicalduration of symptomatic relief from a single intramuscular injection ofbotulinum toxin type A averages about 3 months, although significantlylonger periods of therapeutic activity have been reported.

Although all the botulinum toxins serotypes apparently inhibit releaseof the neurotransmitter acetylcholine at the neuromuscular junction,they do so by affecting different neurosecretory proteins and cleavingthese proteins at different sites. For example, botulinum types A and Eboth cleave the 25 kDa synaptosomal associated protein (SNAP-25), butthey target different amino acid sequences within this protein.Botulinum toxin types B, D, F and G act on vesicle-associated membraneprotein (VAMP, also called synaptobrevin), with each serotype cleavingthe protein at a different site. Finally, botulinum toxin type C1appears to cleave both syntaxin and SNAP-25. These differences inmechanism of action may affect the relative potency and/or duration ofaction of the various botulinum toxin serotypes. Particularly, asubstrate for a botulinum toxin can be found in a variety of differentcell types.

The molecular weight of the botulinum toxin, for all seven of the knownbotulinum toxin serotypes, is about 150 kDa. Interestingly, thebotulinum toxins are released by Clostridial bacterium as complexescomprising the 150 kDa botulinum toxin protein molecule along withassociated non-toxin proteins. Thus, the botulinum toxin type A complexcan be produced by Clostridial bacterium as 900 kDa, 500 kDa or 300 kDaforms. Botulinum toxin types B and C1 are apparently produced as only a700 kDa or 500 kDa complex. Botulinum toxin type D is produced as 300kDa or 500 kDa complexes. Finally, botulinum toxin types E and F areproduced as only approximately 300 kDa complexes. The complexes (i.e.molecular weight greater than about 150 kDa) are believed to contain anon-toxin hemagglutinin proteins, a non-toxin, and non-toxicnon-hemagglutinin protein. These two non-toxin proteins (which alongwith the botulinum toxin molecule comprise the relevant neurotoxincomplex) may act to provide stability against denaturation to thebotulinum toxin molecule and protection against digestive acids when abotulinum toxin is ingested. Additionally, it is possible that thelarger (greater than about 150 kDa molecular weight) botulinum toxincomplexes result in a slower rate of diffusion of the botulinum toxinaway from a site of intramuscular injection of a botulinum toxincomplex.

In vitro studies have indicated that botulinum toxin inhibits potassiumcation-induced release of both acetylcholine and norepinephrine fromprimary cell cultures of brainstem tissue. In addition, it has beenreported that botulinum toxin inhibits the evoked release of bothglycine and glutamate in primary cultures of spinal cord neurons andthat in brain synaptosome preparations botulinum toxin inhibits therelease of each of the neurotransmitters acetylcholine, dopamine,norepinephrine, CGRP, substance P, and glutamate. Thus, when adequateconcentrations are used, the stimulus-evoked release of mostneurotransmitters can be blocked by botulinum toxin.

Botulinum toxin type A can be obtained by establishing and growingcultures of Clostridium botulinum in a fermenter and then harvesting andpurifying the fermented mixture in accordance with known procedures. Allthe botulinum toxin serotypes are initially synthesized as inactivesingle chain proteins which must be cleaved or nicked by proteases tobecome neuroactive. The bacterial strains that make botulinum toxinserotypes A and G possess endogenous proteases and serotypes A and Gcan, therefore, be recovered from bacterial cultures in predominantlytheir active form. In contrast, botulinum toxin serotypes C1, D, and Eare synthesized by nonproteolytic strains and are therefore typicallyinactivated when recovered from culture. Serotypes B and F are producedby both proteolytic and nonproteolytic strains, and thus can berecovered in either the active or inactive form. However, even theproteolytic strains that produce, for example, the botulinum toxin typeB serotype only cleave a portion of the toxin produced. The exactproportion of nicked to unnicked molecules depends on the length ofincubation and the temperature of the culture. Therefore, a certainpercentage of any preparation of, for example, the botulinum toxin typeB toxin is likely to be inactive, possibly accounting for the knownsignificantly lower potency of botulinum toxin type B as compared tobotulinum toxin type A. The presence of inactive botulinum toxinmolecules in a clinical preparation will contribute to the overallprotein load of the preparation, which has been linked to increasedantigenicity, without contributing to its clinical efficacy. Moreover,it is known that botulinum toxin type B has, upon intramuscularinjection, a shorter duration of activity and is also less potent thanbotulinum toxin type A at the same dose level.

High-quality crystalline botulinum toxin type A can be produced from theHall A strain of Clostridium botulinum with characteristics of ≥3×10⁷U/mg, an A260/A278 of less than 0.60 and a distinct pattern of bandingon gel electrophoresis. The known Schantz process can be used to obtaincrystalline botulinum toxin type A. Generally, the botulinum toxin typeA complex can be isolated and purified from an anaerobic fermentation bycultivating Clostridium botulinum type A in a suitable medium. The knownprocess can also be used, upon separation out of the non-toxin proteins,to obtain pure botulinum toxins, such as for example: purified botulinumtoxin type A with an approximately 150 kDa molecular weight with aspecific potency of 1-2×10⁸ LD50 U/mg or greater; purified botulinumtoxin type B with an approximately 156 kDa molecular weight with aspecific potency of 1-2×10⁸ LD50 U/mg or greater, and; purifiedbotulinum toxin type F with an approximately 155 kDa molecular weightwith a specific potency of 1-2×10⁷ LD50 U/mg or greater.

Botulinum toxins and/or botulinum toxin complexes are commerciallyavailable from compound manufacturers known in the art, and purebotulinum toxin can also be used to prepare a pharmaceuticalcomposition.

As with enzymes generally, the biological activities of the botulinumtoxins (which are intracellular peptidases) are dependent, at least inpart, upon their three dimensional conformation. Thus, botulinum toxintype A is detoxified by heat, various chemicals surface stretching andsurface drying. Additionally, it is known that dilution of a botulinumtoxin complex obtained by the known culturing, fermentation andpurification to the very low toxin concentrations used forpharmaceutical composition formulation results in rapid detoxificationof the toxin unless a suitable stabilizing agent is present. Dilution ofthe toxin from milligram quantities to a solution containing nanogramsper milliliter presents significant difficulties because of the rapidloss of specific toxicity upon such great dilution. Since the botulinumtoxin may be used months or years after the toxin containingpharmaceutical composition is formulated, the toxin should be stabilizedwith a suitable stabilizing agent. Thus, as disclosed in the presentinvention, the development of optimal stabilizer technology is necessaryto control the in vivo release of botulinum toxin to a slow releaseform.

It has been reported that botulinum toxin type A has been used inclinical settings as follows:

The usual duration of an intramuscular injection of botulinum toxinadministered in vivo is typically about 3 to 4 months. However, in somecases, botulinum toxin subtype A can have an efficacy for up to 12months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and in somecircumstances for as long as 27 months, when used to treat glands, suchas in the treatment of hyperhydrosis.

In addition to having pharmacologic actions at the peripheral location,botulinum toxins may also have inhibitory effects in the central nervoussystem. Work by Weigand et al, Nauny-Schmiede berg's Arch. Pharmacol.1976; 292, 161-165, and Habermann, Nauny-Schmiedeberg's Arch. Pharmacol.1974; 281, 47-56 showed that botulinum toxin is able to ascend to thespinal area by retrograde transport. As such, a botulinum toxin injectedat a peripheral location, for example intramuscularly, may be retrogradetransported to the spinal cord.

A botulinum toxin has also been proposed for or has been used to treatskin bone and tendon wounds (U.S. Pat. No. 6,447,787); intrathecal pain(see U.S. Pat. No. 6,113,915); various autonomic nerve disorders,including sweat gland disorders (see e.g. U.S. Pat. No. 5,766,605 andGoldman (2000), Aesthetic Plastic Surgery July-August 24(4):280-282);tension headache (U.S. Pat. No. 6,458,365); migraine headache (U.S. Pat.No. 5,714,468); post-operative pain and visceral pain (U.S. Pat. No.6,464,986); hair growth and hair retention (U.S. Pat. No. 6,299,893);psoriasis and dermatitis (U.S. Pat. No. 5,670,484); injured muscles(U.S. Pat. No. 6,423,319); various cancers (U.S. Pat. Nos. 6,139,845 and6,063,768), smooth muscle disorders (U.S. Pat. No. 5,437,291); nerveentrapment syndromes (US Patent Application 2003-0224019); acne (WO03/011333); neurogenic inflammation (U.S. Pat. No. 6,063,768); opticdisorders (see U.S. Pat. No. 6,265,379); pancreatic disorders (see U.S.Pat. Nos. 6,143,306 and 6,261,572); prostate disorders, includingprostatic hyperplasia, prostate cancer and urinary incontinence (seeU.S. Pat. Nos. 6,365,164 and 6,667,041 and Doggweiler R., et alBotulinum toxin type A causes diffuse and highly selective atrophy ofrat prostate, Neurourol Urodyn 1998; 17(4):363); fibromyalgia (U.S. Pat.No. 6,623,742), and piriformis muscle syndrome (see Childers et al.(2002), American Journal of Physical Medicine & Rehabilitation,81:751-759).

U.S. Pat. No. 5,989,545 discloses that a modified clostridial neurotoxinor fragment thereof, preferably a botulinum toxin, chemically conjugatedor recombinantly fused to a particular targeting moiety can be used totreat pain by administration of the agent to the spinal cord.Additionally, it has been disclosed that targeted botulinum toxins (i.e.with a non-native binding moiety) can be used to treat variousconditions (see WO 96/33273; WO 99/17806; WO 98/07864; WO 00/57897; WO01/21213; WO 00/10598).

In addition, a botulinum toxin has been injected into the pectoralmuscle to control pectoral spasm (Senior M., Botox and the management ofpectoral spasm after subpectoral implant insertion, Plastic and ReconSurg, July 2000, 224-225). Controlled release toxin implants are known(see U.S. Pat. Nos. 6,306,423 and 6,312,708) as is transdermal botulinumtoxin administration (U.S. patent application Ser. No. 10/194,805). Itis known that a botulinum toxin can be used to: weaken the chewing orbiting muscle of the mouth so that self inflicted wounds and resultingulcers can be healed (Payne M., et al, Botulinum toxin as a noveltreatment for self mutilation in Lesch-Nyhan syndrome, Ann Neurol 2002September; 52 (3 Supp 1):S157); permit healing of benign cystic lesionsor tumors (Blugerman G., et al., Multiple eccrine hidrocystomas: A newtherapeutic option with botulinum toxin, Dermatol Surg 2003 May;29(5):557-9); treat anal fissure (Jost W., Ten years' experience withbotulinum toxin in anal fissure, Int J Colorectal Dis 2002 September;17(5):298-302); and treat certain types of atopic dermatitis (HeckmannM., et al., Botulinum toxin type A injection in the treatment of lichensimplex: An open pilot study, J Am Acad Dermatol 2002 April;46(4):617-9).

Additionally, a botulinum toxin may have the effect of reducing inducedinflammatory pain in a rat formalin model (Aoki K., et al, Mechanisms ofthe antinociceptive effect of subcutaneous Botox: Inhibition ofperipheral and central nociceptive processing, Cephalalgia 2003September; 23(7):649). Furthermore, it has been reported that botulinumtoxin nerve blockage can cause a reduction of epidermal thickness (Li Y,et al., Sensory and motor denervation influences epidermal thickness inrat foot glabrous skin, Exp Neurol 1997; 147:452-462). Finally, it isknown to administer a botulinum toxin to the foot to treat excessivefoot sweating (Katsambas A., et al., Cutaneous diseases of the foot:Unapproved treatments, Clin Dermatol 2002 November-December;20(6):689-699; Sevim, S., et al., Botulinum toxin-A therapy for palmarand plantar hyperhidrosis, Acta Neurol Belg 2002 December;102(4):167-70), spastic toes (Suputtitada, A., Local botulinum toxintype A injections in the treatment of spastic toes, Am J Phys MedRehabil 2002 October; 81(10):770-5), idiopathic toe walking (Tacks, L.,et al., Idiopathic toe walking: Treatment with botulinum toxin Ainjection, Dev Med Child Neurol 2002; 44(Suppl 91):6), and foot dystonia(Rogers J., et al., Injections of botulinum toxin Ain foot dystonia,Neurology 1993 April; 43(4 Suppl 2)).

Tetanus toxin, as wells as derivatives (i.e. with a non-native targetingmoiety), fragments, hybrids and chimeras thereof can also havetherapeutic utility. The tetanus toxin bears many similarities to thebotulinum toxins. Thus, both the tetanus toxin and the botulinum toxinsare polypeptides made by closely related species of Clostridium(Clostridium tetani and Clostridium botulinum, respectively).Additionally, both the tetanus toxin and the botulinum toxins aredichain proteins composed of a light chain (molecular weight: about 50kDa) covalently bound by a single disulfide bond to a heavy chain(molecular weight: about 100 kDa). Hence, the molecular weight oftetanus toxin and of each of the 7 botulinum toxins (non-complexed) isabout 150 kDa. Furthermore, for both the tetanus toxin and the botulinumtoxins, the light chain bears the domain which exhibits intracellularbiological (protease) activity, while the heavy chain comprises thereceptor binding (immunogenic) and cell membrane translocationaldomains.

Further, both the tetanus toxin and the botulinum toxins exhibit a high,specific affinity for ganglioside receptors on the surface ofpresynaptic cholinergic neurons. Receptor-mediated endocytosis oftetanus toxin in peripheral cholinergic neurons results in retrogradeaxonal transport, blocking the release of inhibitory neurotransmittersfrom central synapses, and causing a spastic paralysis. Contrarily, ithas been believed that receptor-mediated endocytosis of botulinum toxinin peripheral cholinergic neurons hardly results in retrogradetransport, inhibition of acetylcholine exocytosis from the centralsynapses, and a flaccid paralysis. However, very recent report hassuggested that botulinum toxin also can undergo retrograde transportalong axons and possibly inhibit the release of acetylcholine in centralsynapse (Bomba-Warczak et al., Interneuronal Transfer and Distal Actionof Tetanus Toxin and Botulinum Neurotoxins A and Din Central Neurons,Cell Reports, 2016 August; 16, 1974-1987).

Finally, the tetanus toxin and the botulinum toxins resemble each otherin both biosynthesis and molecular architecture. Thus, there is anoverall 34% identity between the protein sequences of tetanus toxin andbotulinum toxin type A, and a sequence identity as high as 62% for somefunctional domains (Binz T. et al., The Complete Sequence of BotulinumNeurotoxin Type A and Comparison with Other Clostridial Neurotoxins, JBiological Chemistry 265(16); 9153-9158:1990).

In one embodiment of the present invention, “acetylcholine” is an esterof choline and acetic acid, which is the first known neurotransmitter.It is distributed throughout neurons, and has a chemical formula ofC₇H₁₆NO₂ and a molecular weight of 146.21 kDa.

Typically, only a single type of small molecule neurotransmitter isreleased by each type of neuron in the mammalian nervous system,although there is evidence which suggests that several neuromodulatorscan be released by the same neuron. The neurotransmitter acetylcholineis secreted by neurons in many areas of the brain, specifically by thelarge pyramidal cells of the motor cortex, several different neurons inthe basal ganglia, the motor neurons that innervate the skeletalmuscles, the preganglionic neurons of the autonomic nervous system (bothsympathetic and parasympathetic), the bag 1 fibers of the muscle spindlefiber, the postganglionic neurons of the parasympathetic nervous system,and some of the postganglionic neurons of the sympathetic nervoussystem. Essentially, only the postganglionic sympathetic nerve fibers tothe sweat glands, the piloerector muscles and a few blood vessels arecholinergic as most of the postganglionic neurons of the sympatheticnervous system secret the neurotransmitter norepinephine. In mostinstances, acetylcholine has an excitatory effect. However,acetylcholine is known to have inhibitory effects at some of theperipheral parasympathetic nerve endings (for example, inhibition ofheart rate by the vagal nerve).

The efferent signals of the autonomic nervous system are transmitted tothe body through either the sympathetic nervous system or theparasympathetic nervous system. The preganglionic neurons of thesympathetic nervous system extend from preganglionic sympathetic neuroncell bodies located in the intermediolateral horn of the spinal cord.The preganglionic sympathetic nerve fibers, extending from the cellbody, synapse with postganglionic neurons located in either aparavertebral sympathetic ganglion or in a prevertebral ganglion. Sincethe preganglionic neurons of both the sympathetic and parasympatheticnervous system are cholinergic, application of acetylcholine to theganglia will excite both sympathetic and parasympathetic postganglionicneurons.

Acetylcholine activates two types of receptors, muscarinic and nicotinicreceptors. The muscarinic receptors are found in all effector cellsstimulated by the postganglionic, neurons of the parasympathetic nervoussystem as well as in those stimulated by the postganglionic cholinergicneurons of the sympathetic nervous system. The nicotinic receptors arefound in the adrenal medulla, as well as within the autonomic ganglia,that is on the cell surface of the postganglionic neuron at the synapsebetween the preganglionic and postganglionic neurons of both thesympathetic and parasympathetic systems. Nicotinic receptors are alsofound in many nonautonomic nerve endings, for example in the membranesof skeletal muscle fibers at the neuromuscular junction.

Acetylcholine is released from cholinergic neurons when small, clear,intracellular vesicles fuse with the presynaptic neuronal cell membrane.A wide variety of non-neuronal secretory cells, such as adrenal medulla(as well as the PC12 cell line) and pancreatic islet cells releasecatecholamines and parathyroid hormone, respectively, from largedense-core vesicles. The PC12 cell line is a clone of ratpheochromocytoma cells extensively used as a tissue culture model forstudies of sympathoadrenal development. Botulinum toxin inhibits therelease of both types of compounds from both types of cells in vitro,when the denervated cells are permeabilized (as by electroporation) ordirectly injected with the toxin. Botulinum toxin is also known to blockrelease of the neurotransmitter glutamate from cortical synaptosomescell cultures.

A neuromuscular junction is formed in skeletal muscle by the proximityof axons to muscle cells. A signal transmitted through the nervoussystem results in an action potential at the terminal axon, withactivation of ion channels and resulting release of the neurotransmitteracetylcholine from intraneuronal synaptic vesicles, for example at themotor endplate of the neuromuscular junction. The acetylcholine crossesthe extracellular space to bind with acetylcholine receptor proteins onthe surface of the muscle end plate. Once sufficient binding hasoccurred, an action potential of the muscle cell causes specificmembrane ion channel changes, resulting in muscle cell contraction. Theacetylcholine is then released from the muscle cells and metabolized bycholinesterases in the extracellular space. The metabolites are recycledback into the terminal axon for reprocessing into further acetylcholine.

In one embodiment of the present invention, “activity of botulinumtoxin” means the potency of toxin. One unit (U) of botulinum toxin isdefined as the amount of botulinum toxin that kills 50% of a group ofmice weighing 18 to 20 g each, when measured by mouse LD50 bioassay(mLD50), a standard assay method. Botulinum toxin, particularlybotulinum toxin serotype A, is the most lethal natural biological agentknown to man, and is 1.8 billion times more lethal than diphtheriatoxin, 600 million times more lethal than sodium cyanide, 30 milliontimes more lethal than cobra toxin and 12 million times more lethal thancholera toxin. Thus, a difference in potency of about 20% in botulinumtoxin results in a significant difference in effect, such as 360 milliontimes diphtheria toxin, or 2.4 million times cholera toxin.

Botulinum toxin formulations got medical or cosmetic purposes aregenerally distributed as lyophilized formulations or liquidformulations, and have a problem in that because botulinum toxin itselfis a protein, the activity thereof becomes very unstable by temperature,pH, light, physical impact, or gas (air, nitrogen, oxygen, etc.). Whenthe potency of botulinum toxin is reduced as described above, it hardlyexhibits its expected effect, and hence it is necessarily required toaccurately predict the potency of botulinum toxin in a preparation stepor a use step.

In one embodiment of the present invention, “antibody” is a term knownin the art and refers to a specific protein molecule that is directedagainst an antigenic site. For the purpose of the present invention, theantibody means an antibody that binds specifically to SNAP25 protein.This antibody may be produced according to a conventional method. Theantibodies of the present invention include a partial peptide that maybe produced from the protein, and the partial peptide of the presentinvention comprises at least 7 amino acids, preferably at least 9 aminoacids, more preferably at least 12 amino acids. The form of antibodyaccording to the present invention is not specifically limited, andpolyclonal antibodies, monoclonal antibodies, or portions thereof whichhave antigen binding ability are included in the antibodies of thepresent invention, and all immunoglobulin antibodies are included in theantibodies of the present invention. Furthermore, the antibodies of thepresent invention also include special antibodies such as humanizedantibodies. The antibodies of the present invention include not only acomplete antibody having light chains and heavy chains, but also afunctional fragment of the antibody molecule. The expression “functionalfragment of the antibody molecule” refers to a fragment having at leastantigen binding ability, and examples of the functional fragment includeFab, F(ab′), F(ab′)2, Fv and the like.

In one embodiment of the present invention, the term “kit” means a setof compositions and accessories required for a specific purpose. Withthe respect of the purpose of the present invention, the kit of thepresent invention comprises either an antibody that binds specificallyto SNAP25_(FL) or SNAP25₁₉₇, or a composition containing the antibody,or a cell culture dish coated with the antibody, in order to measure theactivity of botulinum toxin.

In one embodiment of the present invention, there is provided a cellline, clonally selected from Neuro-2a which is a parental neuronal cellline.

The present inventors have selected Neuro-2a cells having sensitivity tobotulinum toxin, which is similar to that of the SiMa cell line, from 13different neuronal cell lines, and finally selected N2-42F (accessionnumber: KCTC 13712BP), which consistently shows high sensitivity tobotulinum toxin, from the Neuro-2a cells through a clonal selectionprocess, thereby completing the present invention.

The cell line of the present invention may be used to determine theactivity of botulinum toxin or to detect botulinum toxin.

The cell line of the present invention maintains its sensitivity tobotulinum toxin even if the passage continues, and thus can be used verysuitably in a cell-based assay platform.

The cell line for determining the activity of botulinum toxin accordingto the present invention means homogeneous single cells isolated from aparental neuronal cell line corresponding to a population includingvarious types of cells, and refers to cells having common geneticfeatures, for example, high or low gene expression levels of a specificgene, or the like.

The cell line for determining the activity of botulinum toxin may beisolated from the parental neuronal cell line through a method such asclonal selection, or produced by regulating the expression levels of thegenes. Regulation of the expression levels of the genes can be achievedby a conventional method for regulating gene expression, for example,transformation, promoter manipulation, or the like.

The parental neuronal cell line of the present invention may include anyimmortalized cell line derived from nerve, and may be preferablyNeuro-2a cells, more preferably Neuro-2a cells (accession number: KCTCAC28106), but is not limited thereto. The Neuro-2a cells are murineneuronal cells that may generally be used to measure LD50, and thedoubling time is 34 to 100 hours in conventional SiMa cells which areused to determine the activity of botulinum toxin, but is only 24 hoursin the Neuro-2a cells, indicating that the Neuro-2a cells are verysuitable not only for cell-based determination of the activity ofbotulinum toxin, but also for detection of botulinum toxin. Furthermore,the Neuro-2a cells correspond to a population containing various typesof cells when observed with a microscope, and thus have the advantage ofbeing very suitable for selection of only single cells more sensitive tobotulinum toxin therefrom.

The cell line of the present invention, clonally selected from theparental neuronal cell line Neuro-2a, may be N2-42F (accession number:KCTC 13712BP), but is not limited thereto.

The cell line of the present invention for determining the activity ofbotulinum toxin may be used to detect botulinum toxin or determine theactivity thereof. The cell line is sensitive to botulinum toxin, andthus can detect the presence or absence of botulinum toxin in a sampleof interest, and can also measure the degree of toxicity of botulinumtoxin depending on the concentration of botulinum toxin.

The botulinum toxin of the present invention is a neurotoxic proteinproduced by the bacterium Clostridium botulinum, and can be classifiedinto a total of seven serotypes: A, B, C (C1, C2), D, E, F and G. Thebotulinum toxin affects different neurosecretory proteins depending onthe serotypes and cleaves these proteins at different sites.Specifically, both botulinum toxin serotypes A and E can cleave SNAP25(synaptosomal nerve-associated protein 25), and botulinum toxinserotypes B, D, F and G can cleave VAMP (vesicle-associated membraneprotein), and botulinum toxin serotype C1 can cleave both syntaxin andSNAP25, thereby inducing neurotoxicity. Preferably, the botulinum toxinmay be botulinum toxin serotype A or botulinum toxin serotype B, morepreferably botulinum toxin serotype A, but is not limited thereto.

Since the botulinum toxin serotypes A and B of the present invention arepurified and widely used for treatment of dystonia, aestheticapplications, and the like, and thus when the cell line of the presentinvention for determining the activity of botulinum toxin is used tomeasure the potency of botulinum toxin, there is an advantage in thatthe concentration at which side effects can occur can be determined,thereby solving the problems which can occur when the botulinum toxin isused for the above-described applications.

In another embodiment of the present invention, there is provided acell-based method for determining the activity of botulinum toxin.

The method of the present invention comprises the steps of: culturingthe cell line according to the present invention; treating the culturedcell line with the botulinum toxin; and measuring the sensitivity of thebotulinum toxin-treated cell line to the botulinum toxin.

The cell-based method for determining the activity of botulinum toxin ofthe present invention can be achieved by treating the cell line fordetermining the activity of the botulinum toxin with the botulinumtoxin, and measuring the sensitivity of the cell line to the botulinumtoxin. Thus, the description of the contents related to the cell linefor determining the activity of the botulinum toxin, the botulinumtoxin, the neurosecretory proteins cleaved by the botulinum toxin, theparental neuronal cell line, and the like, will be omitted in order toavoid excessive complexity of the specification due to repeateddescription thereof.

In the step of culturing the cell line in the present invention, theculturing of the cell line may be performed in a culture plate coatedwith poly-D-lysine. When the plate coated with poly-D-lysine is used,the cell line according to the present invention can be distributeduniformly, attached firmly, and maintained at a healthy cell state,compared to when using a plate which is generally used for culturing ofa cell line or a plate coated with gelatin or collagen.

The step of measuring the sensitivity of the cell line to the botulinumtoxin in the present invention may comprise measuring the cleavage ofendogeneous neurosecretory protein caused by the botulinum toxin.Specifically, in the case of botulinum toxin serotype A and serotype E,the cleavage of SNAP25 may be measured, and in the case of botulinumtoxin serotypes B, D, F and G, the cleavage of VAMP may be measured, andin the case of botulinum toxin serotype C1, the cleavage of syntaxinand/or SNAP25 may be measured.

The measurement of the cleavage in the present invention may be achievedthrough a method of detecting a protein using an antibody specific forthe cleaved peptide of the endogeneous neurosecretory protein, or thelike.

The antibody of the present invention means a protein molecule that canrecognizes the whole or cleaved peptide of the neurosecretory protein asan antigen and can bind specifically to the neurosecretory protein, andexamples thereof include polyclonal antibodies, monoclonal antibodies,and recombinant antibodies.

The method of detecting the protein, which is used in the presentinvention, may be any conventional method for detecting protein, andexamples thereof include, but are not limited to, Western blottingassay, ELISA (enzyme linked immunosorbent assay), RIA(radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion,rocket immunoelectrophoresis, immunohistostaining, immunoprecipitationassay, complement fixation assay, fluorescence activated cell sorter(FACS), protein chip assay, and the like.

In still another embodiment of the present invention, there is provideda cell-based method for detecting botulinum toxin.

The method of the present invention comprises the steps of: culturingthe cell line according to the present invention; treating the culturedcell line with a sample of interest; and measuring the sensitivity ofthe sample-treated cell line to the botulinum toxin.

The cell-based method for detecting the botulinum toxin according to thepresent invention may be achieved by treating the cell line with thesample of interest instead of the botulinum toxin used in the cell-basedmethod for determining the activity of the botulinum toxin, andmeasuring the sensitivity of the cell line to the botulinum toxin. Thus,the description of the contents related to the cell line for determiningthe activity of the botulinum toxin, the botulinum toxin, theneurosecretory proteins cleaved by the botulinum toxin, the parentalneuronal cell line, the coated plate, the protein detection method, theantibody, and the like, will be omitted in order to avoid excessivecomplexity of the specification due to repeated description thereof.

The sample of interest which is used in the present invention is asample expected to contain botulinum toxin, and examples thereof mayinclude biological samples, including cell culture supernatants, blood,saliva, sputum, cerebrospinal fluids, secretions, lymphatic fluids,dialysis fluids, body fluids, urine and the like, and chemical samplescontaining compounds.

In still another embodiment of the present invention, there is providedan antibody that binds specifically to SNAP25, wherein the SNAP25 isSNAP25_(FL) or SNAP25₁₉₇, and the antibody comprises: a heavy-chain CDR1region which is any one selected from the group consisting of SEQ IDNOs: 11 to 13, 28 to 33, and 55 to 56; a heavy-chain CDR2 region whichis any one selected from the group consisting of SEQ ID NOs: 14 to 16,34 to 39, and 57 to 58; a heavy-chain CDR3 region which is any oneselected from the group consisting of SEQ ID NOs: 17 to 19, 40 to 46,and 59 to 60; a light-chain CDR1 region which is any one selected fromthe group consisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62; alight-chain CDR2 region which is any one selected from the groupconsisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and alight-chain CDR3 region which is any one selected from the groupconsisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to 66.

More specifically, the antibody is preferably an antibody that bindsspecifically to SNAP25_(FL) and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 11; a heavy-chain CDR2 region represented bySEQ ID NO: 14; a heavy-chain CDR3 region represented by SEQ ID NO: 17; alight-chain CDR1 region represented by SEQ ID NO: 20; a light-chain CDR2region represented by SEQ ID NO: 23; and a light-chain CDR3 regionrepresented by SEQ ID NO: 25. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 83 and 84, but is not limitedthereto.

Moreover, the antibody is preferably an antibody that binds specificallyto SNAP25_(FL) and comprises: a heavy-chain CDR1 region represented bySEQ ID NO: 12; a heavy-chain CDR2 region represented by SEQ ID NO: 15; aheavy-chain CDR3 region represented by SEQ ID NO: 18; a light-chain CDR1region represented by SEQ ID NO: 21; a light-chain CDR2 regionrepresented by SEQ ID NO: 24; and a light-chain CDR3 region representedby SEQ ID NO: 26. More specifically, the antibody may be an antibodyrepresented by SEQ ID NOs: 87 and 88, but is not limited thereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25_(FL) and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 13; a heavy-chain CDR2 region represented bySEQ ID NO: 16; a heavy-chain CDR3 region represented by SEQ ID NO: 19; alight-chain CDR1 region represented by SEQ ID NO: 22; a light-chain CDR2region represented by SEQ ID NO: 24; and a light-chain CDR3 regionrepresented by SEQ ID NO: 27. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 89 and 90, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 28; a heavy-chain CDR2 region represented bySEQ ID NO: 34; a heavy-chain CDR3 region represented by SEQ ID NO: 40; alight-chain CDR1 region represented by SEQ ID NO: 47; a light-chain CDR2region represented by SEQ ID NO: 50; and a light-chain CDR3 regionrepresented by SEQ ID NO: 52. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 71 and 72, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 29; a heavy-chain CDR2 region represented bySEQ ID NO: 35; a heavy-chain CDR3 region represented by SEQ ID NO: 41; alight-chain CDR1 region represented by SEQ ID NO: 48; a light-chain CDR2region represented by SEQ ID NO: 50; and a light-chain CDR3 regionrepresented by SEQ ID NO: 52. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 73 and 74, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 29; a heavy-chain CDR2 region represented bySEQ ID NO: 36; a heavy-chain CDR3 region represented by SEQ ID NO: 42; alight-chain CDR1 region represented by SEQ ID NO: 47; a light-chain CDR2region represented by SEQ ID NO: 50; and a light-chain CDR3 regionrepresented by SEQ ID NO: 52. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 75 and 76, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 33; a heavy-chain CDR2 region represented bySEQ ID NO: 35; a heavy-chain CDR3 region represented by SEQ ID NO: 43; alight-chain CDR1 region represented by SEQ ID NO: 48; a light-chain CDR2region represented by SEQ ID NO: 50; and a light-chain CDR3 regionrepresented by SEQ ID NO: 52. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 77 and 78, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 30; a heavy-chain CDR2 region represented bySEQ ID NO: 37; a heavy-chain CDR3 region represented by SEQ ID NO: 44; alight-chain CDR1 region represented by SEQ ID NO: 48; a light-chain CDR2region represented by SEQ ID NO: 50; and a light-chain CDR3 regionrepresented by SEQ ID NO: 52. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 79 and 80, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 31; a heavy-chain CDR2 region represented bySEQ ID NO: 38; a heavy-chain CDR3 region represented by SEQ ID NO: 45; alight-chain CDR1 region represented by SEQ ID NO: 47; a light-chain CDR2region represented by SEQ ID NO: 50; and a light-chain CDR3 regionrepresented by SEQ ID NO: 53. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 81 and 82, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25₁₉₇ and comprises: a heavy-chain CDR1 regionrepresented by SEQ ID NO: 32; a heavy-chain CDR2 region represented bySEQ ID NO: 39; a heavy-chain CDR3 region represented by SEQ ID NO: 46; alight-chain CDR1 region represented by SEQ ID NO: 49; a light-chain CDR2region represented by SEQ ID NO: 51; and a light-chain CDR3 regionrepresented by SEQ ID NO: 54. More specifically, the antibody may be anantibody represented by SEQ ID NOs: 85 and 86, but is not limitedthereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25_(FL) and SNAP25₁₉₇ and comprises: a heavy-chainCDR1 region represented by SEQ ID NO: 55; a heavy-chain CDR2 regionrepresented by SEQ ID NO: 57; a heavy-chain CDR3 region represented bySEQ ID NO: 59; a light-chain CDR1 region represented by SEQ ID NO: 61; alight-chain CDR2 region represented by SEQ ID NO: 63; and a light-chainCDR3 region represented by SEQ ID NO: 65. More specifically, theantibody may be an antibody represented by SEQ ID NOs: 67 and 68, but isnot limited thereto.

In addition, the antibody is preferably an antibody that bindsspecifically to SNAP25_(FL) and SNAP25₁₉₇ and comprises: a heavy-chainCDR1 region represented by SEQ ID NO: 56; a heavy-chain CDR2 regionrepresented by SEQ ID NO: 58; a heavy-chain CDR3 region represented bySEQ ID NO: 60; a light-chain CDR1 region represented by SEQ ID NO: 62; alight-chain CDR2 region represented by SEQ ID NO: 64; and a light-chainCDR3 region represented by SEQ ID NO: 66. More specifically, theantibody may be an antibody represented by SEQ ID NOs: 69 and 70, but isnot limited thereto.

In still another embodiment of the present invention, there is providedan antibody composition comprising the antibody, or a culture dishcoated with the antibody, or a kit comprising the antibody compositionor the culture dish.

In still another embodiment of the present invention, there is provideda hybridoma cell capable of producing the antibody, wherein thehybridoma cell is a fusion of a spleen cell and myeloma cell of a mouseinjected with any one or more peptides selected from the groupconsisting of SEQ ID NOs: 1 to 10.

In still another embodiment of the present invention, there is provideda method for determining the activity of botulinum toxin, comprising thesteps of: (a) treating a neuronal cell with the botulinum toxin; and (b)measuring SNAP25_(FL) or SNAP25₁₉₇ in the neuronal cell by any one ormore antibodies selected from among antibodies represented by SEQ IDNOs: 67 to 90, wherein the botulinum toxin is botulinum toxin type A.

In still another embodiment of the present invention, there is provideda method for detecting botulinum toxin, comprising the steps of: (a)treating a neuronal cell with a sample of interest; (b) measuringSNAP25₁₉₇ in the neuronal cell by any one or more antibodies selectedfrom among antibodies represented by SEQ ID NOs: 67 to 82, SEQ ID NO:85, or SEQ ID NO: 86; and (c) determining that when SNAP25₁₉₇ ismeasured, the botulinum toxin is present in the sample, wherein thebotulinum toxin is botulinum toxin type A.

In still another embodiment of the present invention, there is provideda cell-based method for analyzing the potency of neurotoxin, comprisingthe steps of: (a) culturing a Neuro-2a-derived neuronal cell line; (b)treating the neuronal cell line with the neurotoxin; (c) treating theneuronal cell line or a sample, obtained from the neuronal cell line,with an antibody that binds specifically to SNAP25_(FL) and SNAP25₁₉₇;and (d) treating the neuronal cell line of step (c) with an antibodythat binds specifically to SNAP25₁₉₇ without binding to SNAP25_(FL),wherein the Neuro-2a-derived cell line is a N2-42F cell line (accessionnumber: KCTC 13712BP), the neurotoxin is botulinum toxin, and theneurotoxin in step (b) is diluted with a medium containing GT1b(ganglioside GT1b trisodium salt) and used to treat the cell line.

The antibody used in step (c) of the cell-based method for analyzing thepotency of the neurotoxin comprises: a heavy-chain CDR1 regionconsisting of SEQ ID NO: 55 or 56; a heavy-chain CDR2 region consistingof SEQ ID NO: 57 or 58; a heavy-chain CDR3 region consisting of SEQ IDNO: 59 or 60; a light-chain CDR1 region consisting of SEQ ID NO: 61 or62; a light-chain CDR2 region consisting of SEQ ID NO: 63 or 64; and alight-chain CDR3 region consisting of SEQ ID NO: 65 or 66. Morespecifically, the antibody used in step (c) of the cell-based method foranalyzing the potency of the neurotoxin comprises: a heavy-chain CDR1region composed of SEQ ID NO: 55; a heavy-chain CDR2 region consistingof SEQ ID NO: 57; a heavy-chain CDR3 region consisting of SEQ ID NO: 59;a light-chain CDR1 region consisting of SEQ ID NO: 61; a light-chainCDR2 region consisting of SEQ ID NO: 63; and a light-chain CDR3 regionconsisting of SEQ ID NO: 65. In addition, the antibody used in step (c)of the cell-based method for analyzing the potency of the neurotoxincomprises: a heavy-chain CDR1 region consisting of SEQ ID NO: 56; aheavy-chain CDR2 region consisting of SEQ ID NO: 58; a heavy-chain CDR3region consisting of SEQ ID NO: 60; a light-chain CDR1 region consistingof SEQ ID NO: 62; a light-chain CDR2 region consisting of SEQ ID NO: 64;and a light-chain CDR3 region consisting of SEQ ID NO: 66.

In addition, the antibody used in step (d) of the cell-based method foranalyzing the potency of the neurotoxin comprises: a heavy-chain CDR1region composed of any one selected from the group consisting of SEQ IDNOs: 28 to 33; a heavy-chain CDR2 region composed of any one selectedfrom the group consisting of SEQ ID NOs: 34 to 39; a heavy-chain CDR3region composed of any one selected from the group consisting of SEQ IDNOs: 40 to 46; a light-chain CDR1 region composed of any one selectedfrom the group consisting of SEQ ID NOs: 47 to 49; a light-chain CDR2region composed of any one selected from the group consisting of SEQ IDNOs: 50 to 51; and a light-chain CDR3 region composed of any oneselected from the group consisting of SEQ ID NOs: 52 to 54. Morespecifically, the antibody used in step (d) of the cell-based method foranalyzing the potency of the neurotoxin comprises: a heavy-chain CDR1region consisting of SEQ ID NO: 28; a heavy-chain CDR2 region consistingof SEQ ID NO: 34; a heavy-chain CDR3 region consisting of SEQ ID NO: 40;a light-chain CDR1 region consisting of SEQ ID NO: 47; a light-chainCDR2 region consisting of SEQ ID NO: 50; and a light-chain CDR3 regionconsisting of SEQ ID NO: 52. In addition, the antibody used in step (d)of the cell-based method for analyzing the potency of the neurotoxincomprises: a heavy-chain CDR1 region consisting of SEQ ID NO: 29; aheavy-chain CDR2 region consisting of SEQ ID NO: 35; a heavy-chain CDR3region consisting of SEQ ID NO: 41; a light-chain CDR1 region consistingof SEQ ID NO: 48; a light-chain CDR2 region consisting of SEQ ID NO: 50;and a light-chain CDR3 region consisting of SEQ ID NO: 52. In addition,the antibody used in step (d) of the cell-based method for analyzing thepotency of the neurotoxin comprises: a heavy-chain CDR1 regionconsisting of SEQ ID NO: 29; a heavy-chain CDR2 region consisting of SEQID NO: 36; a heavy-chain CDR3 region consisting of SEQ ID NO: 42; alight-chain CDR1 region consisting of SEQ ID NO: 47; a light-chain CDR2region consisting of SEQ ID NO: 50; and a light-chain CDR3 regionconsisting of SEQ ID NO: 52. Furthermore, the antibody used in step (d)of the cell-based method for analyzing the potency of the neurotoxincomprises: a heavy-chain CDR1 region consisting of SEQ ID NO: 33; aheavy-chain CDR2 region consisting of SEQ ID NO: 35; a heavy-chain CDR3region consisting of SEQ ID NO: 43; a light-chain CDR1 region consistingof SEQ ID NO: 48; a light-chain CDR2 region consisting of SEQ ID NO: 50;and a light-chain CDR3 region consisting of SEQ ID NO: 52. Moreover, theantibody used in step (d) of the cell-based method for analyzing thepotency of the neurotoxin comprises: a heavy-chain CDR1 regionconsisting of SEQ ID NO: 30; a heavy-chain CDR2 region consisting of SEQID NO: 37; a heavy-chain CDR3 region consisting of SEQ ID NO: 44; alight-chain CDR1 region consisting of SEQ ID NO: 48; a light-chain CDR2region consisting of SEQ ID NO: 50; and a light-chain CDR3 regionconsisting of SEQ ID NO: 52. In addition, the antibody used in step (d)of the cell-based method for analyzing the potency of the neurotoxincomprises: a heavy-chain CDR1 region consisting of SEQ ID NO: 31; aheavy-chain CDR2 region consisting of SEQ ID NO: 38; a heavy-chain CDR3region consisting of SEQ ID NO: 45; a light-chain CDR1 region consistingof SEQ ID NO: 47; a light-chain CDR2 region consisting of SEQ ID NO: 50;and a light-chain CDR3 region consisting of SEQ ID NO: 53. In addition,the antibody used in step (d) of the cell-based method for analyzing thepotency of the neurotoxin comprises: a heavy-chain CDR1 regionconsisting of SEQ ID NO: 32; a heavy-chain CDR2 region consisting of SEQID NO: 39; a heavy-chain CDR3 region consisting of SEQ ID NO: 46; alight-chain CDR1 region consisting of SEQ ID NO: 49; a light-chain CDR2region consisting of SEQ ID NO: 51; and a light-chain CDR3 regionconsisting of SEQ ID NO: 54.

In still another embodiment of the present invention, there is provideda cell culture medium for treating a neuronal cell with neurotoxin,containing GT1b (ganglioside GT1b trisodium salt). The concentration ofGT1b in the cell culture medium may be 25 to 75 μg/ml, and the cellculture medium further contains creatine and arginine. Furthermore, theconcentration of creatine in the cell culture medium may be 0.1 to 10mM, and the concentration of arginine in the cell culture medium may be0.5 to 50 mM. In addition, the cell culture medium may be RPMI 1640(Roswell Park Memorial Institute 1640) medium.

Hereinafter, each step of the present invention will be described indetail.

Advantageous Effects

Recently, there has been a rapid increase in the demand for botulinumtoxin for medical and cosmetic purposes, but there is no stable andreproducible cell-based assay method for measuring the potency ofbotulinum toxin. Because botulinum toxin is a very potent neurotoxinprotein, the development of highly specific and sensitive cells andantibodies is particularly required for accurate cell-based measurementof the potency of the botulinum toxin.

The present invention relates to an antibody for determining theactivity of botulinum toxin and an antibody composition comprising thesame. A novel cell line according to the present invention has asignificantly short doubling time compared to conventional SiMa cellswhich are used to determine the activity of botulinum toxin or to detectbotulinum toxin, and also has significantly high sensitivity tobotulinum toxin compared to the parental cell line, indicating that itis very suitable for cell-based determination or detection of theactivity of botulinum toxin. Furthermore, the cell line according to thepresent invention can be attached to and cultured stably in a culturedish coated with poly-d-lysine (PDL), and thus can be very effectivelyused for cell-based determination or detection of the activity ofbotulinum toxin.

An anti-botulinum toxin antibody according to the present invention is amonoclonal antibody having binding specificity for {circle around (1)}SNAP25_(FL), {circle around (2)} SNAP25₁₉₇, or {circle around (3)}SNAP25_(FL) and SNAP25₁₉₇, and has significantly excellent specificityand sensitivity. Thus, it is expected to be actively used in thepharmaceutical and cosmetic fields.

Moreover, the present invention relates to an optimal cell-based potencyassay (CBPA) which uses N2-42F cells and a monoclonal antibody having asignificantly high binding affinity and specificity for SNAP25, and thisCBPA can measure the potency of 0.5 U or less of botulinum toxin. TheCBPA employing the cells and antibody of the present invention isexpected to become a highly reliable and reproducible cell-based potencyassay for botulinum toxin.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of Western blot analysis performed to measuresensitivity to botulinum toxin A (BoNT/A) in neuronal cells according toone example of the present invention. Lane M represents a protein sizemarker; lane 1 represents the expression level of SNAP25 protein in atotal cell lysate not intoxicated with BoNT/A; and lane 2 represents theexpression level of SNAP25 protein in a total cell lysate intoxicatedwith BoNT/A. In addition, N2a represents Neuro-2a cells, and K-BM1represents KP-N-RT-BM-1 cells.

FIG. 2 shows the results of Western blot analysis performed to examinethe degree of cleavage of SNAP25 confirmed in a three-step clonalselection process according to one example of the present invention. In2^(nd) clonal selection, only clone 42 showing the significant cleavageof SNAP25 caused by BoNT/A was selected from among 6 clones includingclone 42, and in 3^(rd) clonal selection, clone 24 (42F) consistentlyshows the cleavage of SNAP25 by BoNT/A in a plurality of the sameexperiments.

FIG. 3 is a graph showing the results of measuring the doubling timebetween N2-42F cells and their parental Neruo-2a cells according to oneexample of the present invention.

FIG. 4 shows 20× images of Neruo-2a (which is a parental cell line),SiMa cells and clone N2-42F imaged using Leica DMi8, when reached 60%confluence, in order to confirm the morphology of the cells according toone example of the present invention.

FIG. 5 shows images of N2-42F cultured in plates coated with each ofcollagen type IV, gelatin and poly-D-lysine, according to one example ofthe present invention.

FIGS. 6a and 6b show the results of Western blot analysis performed toexamine the degree of cleavage of SNAP25, which appears when SiMa cellsand N2-42F were treated with various concentrations of BoNT/A, accordingto one example of the present invention, and the degree of cleavage ofSANP25 or Vamp2, which appears when N2-42F and Neuro-2a were treatedwith various types of botulinum toxin.

FIGS. 7a and 7b shows the results of Western blot analysis performed toexamine the passage stability of N2-42F obtained through the clonalselection process of the present invention, according to one example ofthe present invention.

FIG. 8 is a schematic view showing the positions of SNAP25 antigenpeptides for producing a monoclonal or polyclonal antibody usingsynthetic peptides according to one example of the present invention.

FIG. 9 is a schematic view showing a process for forming hybridoma cellsfor producing a monoclonal antibody, and a process for screening clones,according to one example of the present invention.

FIGS. 10a and 10b show the results of initially screening hybridomacells using ELISA in order to produce a monoclonal antibody according toone example of the present invention.

FIGS. 11a to 11c show the results of re-screening cells for single-cellclone production from the initial hybridoma cell screening resultsaccording to one example of the present invention.

FIGS. 12a to 12c show the results of second re-selection for producingsingle-cell clones according to one example of the present invention.

FIGS. 13a to 13c show the results of third re-selection for producingsingle-cell clones according to one example of the present invention.

FIGS. 14a and 14b shows the pattern of IgG isolated from rabbit serumprotein for producing a polyclonal antibody according to one example ofthe present invention. In FIG. 14a , lane M represents a size marker;lane 1 represents flow-through; lane 2 represents a pool of eluted IgGat pH 5.5; lane 3 represents a pool of eluted IgG at pH 4.0; lane 4represents a pool of eluted IgG at pH 2.5; and lane 5 represents a poolof eluted IgG at pH 11.5.

FIGS. 15a and 15b show the results of kinetic analysis of monoclonalantibodies produced in the present invention, according to one exampleof the present invention. In FIG. 15, IgGs loaded on AMC biosensorsinclude C16 IgG (I), C24 IgG (II), C4 IgG (III), and C7 IgG (IV); arepresents antibody loading; b represents washing; c representsassociation of antigen; and d represents dissociation of antigen.

FIGS. 16a and 16b show the results of kinetic analysis of monoclonalantibodies, produced in the present invention and associated with anddissociated from serially diluted recombinant GST-SNAP25, according toone example of the present invention. In FIG. 16, IgGs loaded on AMCbiosensors include C14 (I), C24 (II), D2 (III), D6 (IV), E6 (V), and A15(VI); a represents antibody loading; b represents association ofantigen; and c represents dissociation of antigen.

FIG. 17 shows the results of Western blot analysis performed to examinethe antigen binding specificity of monoclonal antibodies produced in thepresent invention, according to one example of the present invention. InFIG. 17, lane 1 represents SNAP25_(FL), and lane 2 representsGST-SNAP25₁₉₇.

FIGS. 18a and 18b show the results of Western blot analysis performed toexamine the antigen binding specificity of a monoclonal antibody,produced in the present invention and conjugated with HRP, according toone example of the present invention. In FIG. 18a , lane M represents asize marker; lane 1 represents unconjugated C16 IgG (9 mg); lane 2represents activated HRP (4 mg); lane 3 represents C16 IgG/HRP mixture(C16 IgG-HRP) before incubation (4.5 mg); lane 4 represents C16 IgG-HRPafter incubation (4.5 mg); lane 5 represents C16 IgG-HRP after blocking(4.3 mg); lane 6 represents C16 IgG-HRP after removal of free HRP bydialysis (4.3 mg); and a represents C16 IgG-HRP conjugate.

FIGS. 19a to 19c show the results of SDS-PAGE electrophoresis of amonoclonal antibody, produced in the present invention and conjugatedwith biotin, according to one example of the present invention. In FIGS.19a and 19b , lane M represents a size marker; lane 1 represents A15 IgGalone; lane 2 represents A15 IgG conjugated with 0.1 mM biotin; lane 3represents A15 IgG conjugated with 0.25 mM biotin; and lane 4 representsA15 IgG conjugated with 0.5 mM biotin.

FIGS. 20a and 20b shows the results of SDS-PAGE electgrophoresis of apolyclonal antibody, produced in the present invention and crosslinkedwith AP, according to one example of the present invention. In FIG. 20a, lane M represents a size marker; lane 1 represents unconjugated IgG;lane 2 represents AP; lane 3 represents AP-IgG conjugates; a representsthe stacking gel portion of polyacryamide gel; and b represents AP-IgGconjugates.

FIGS. 21a to 21c show the results of optimizing intoxidation time in amethod of determining the activity of botulinum toxin, according to oneexample of the present invention.

FIG. 22 shows the results of optimizing intoxidation medium in a methodof determining the activity of botulinum toxin, according to one exampleof the present invention.

FIGS. 23a and 23b show the results of optimizing a sensitizer in amethod of determining the activity of botulinum toxin, according to oneexample of the present invention.

FIGS. 24a to 24c show the results of optimizing GT1b in a method ofdetermining the activity of botulinum toxin, according to one example ofthe present invention.

FIGS. 25a and 25b show the results of optimizing N2/B27 in a method ofdetermining the activity of botulinum toxin, according to one example ofthe present invention.

FIGS. 26a and 26b show the results of optimizing capture antibodytreatment in a method of determining the activity of botulinum toxin,according to one example of the present invention.

FIGS. 27a and 27b show the results of optimizing detection antibodytreatment in a method of determining the activity of botulinum toxin,according to one example of the present invention.

FIG. 28 shows the results of optimizing a method of detecting theactivity of HRP conjugates in a method of determining the activity ofbotulinum toxin, according to one example of the present invention.

FIG. 29 is a schematic view showing a sandwich ELISA method fordetermining the activity of botulinum toxin according to one example ofthe present invention.

FIGS. 30a to 30c show the results of examining the accuracy andlinearity of a sandwich ELISA method for determining the activity ofbotulinum toxin, according to one example of the present invention.

FIGS. 31a to 31c show the results of measuring bio-potency by a sandwichELISA method for determining the activity of botulinum toxin, accordingto one example of the present invention.

MODE FOR INVENTION

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to those skilled in theart that these examples are for illustrative purposes only and are notintended to limit the scope of the present invention.

Materials and Method 1: Reagents for Cell Development

0.25% trypsin EDTA (GIBCO™ 25200056), 12-well plate (Corning CLS3513),24-well plate (Falcon 353047), 6-well plate (Falcon 353046), antibioticantimycotic solution (AA) (100×) (Sigma A5955), boric acid (SigmaB6768), collagen from human placenta (Sigma C5533), DMSO (Sigma D2650),DPBS (Welgene LB001-02), DTT (Sigma D0632), fetal bovine serum (FBS) (YIFrontier US-FBS-500), gelatin solution (Sigma G1393), GlutaMAX™ (GIBCO™35050061), glycerol (Affymatrix USB 16374), MEM (GIBCO™ 11095080), MEMnon-essential amino acid (NEAA) (100×) (GIBCO™ 11140050), PCR MycoplasmaDetection Set (Takara Bio 6601), poly-D-lysine hydrobromide (SigmaP6407), polysorbate (Sigma P7949), RIPA buffer (10×) (abcam ab156034),sodium pyruvate (GIBCO™ 11360070), sodium tetraborate (Sigma 221732),T75 flask (Falcon BD353136), TAKARA EX TAQ™ (Takara Bio RR001), TRYPLE™Express Enzyme (1×) (GIBCO™ 12604021).

Materials and Method 2. Preparation of Botulinum Toxin A Stock Solution,Diluent, and BoNT/A Toxic Medium

Purified botulinum toxin serotype A(BoNT/A) was provided by Hugel(EXBII1501).

Materials and Method 2-1. Preparation of BoNT/A Working Stock Solution

Purified BoNT/A was provided by Hugel (EXBII1501). BoNT/A was diluted tomake working stock solution (10 nM) using a toxin dilution bufferconsisting of 50 mM sodium phosphate, pH 7.0, 1 mM DTT, 0.05%polysorbate, 20% glycerol, and 0.2 mg/ml of acetylated-BSA. BoNT/Aworking stocks were stored in aliquots at −80° C. prior to use. AndBoNT/A stock solution was prepared as follows.

(1) Master Stock (200 U/ml): Re-suspend the lyophilized BoNT/A (200 U)in 1 ml of intoxication medium or saline solution, and leave it at RTfor 10 min.(2) Stock A (50 U/ml): Aliquot 150 μl of the master stock in a sterilemicrofuge tube with 450 μl of intoxication medium at RT (i.e., 1:4dilution of master stock).(3) Stock B (5 U/ml): Aliquot 20 μl of the stock A in a sterilemicrofuge tube with 180 μl of intoxication medium at RT (i.e., 1:10dilution of stock A).(4) Stock C (0.5 U/ml): Aliquot 20 μl of the stock B in a tubecontaining 180 μl of intoxication medium at RT (1:10 dilution of stockB).

Materials and Method 2-2. Sample Preparation for Standard Curve

BoNT/A Standard Reference Samples for Standard Curve prepared asfollows.1. Standard Stock A (50 pM, 113.6 U/ml): Re-suspend the lyophilizedBoNT/A (100 U) in 880 μl of intoxication medium or saline solution, andleave it at RT for 10 min.2. Standard Stock B (10 pM, 22.7 U/ml): Aliquot 50 μl of Stock A in asterile microfuge tube with 200 μl of intoxication medium at RT (i.e.,1:5 dilution of Stock A).3. Standard Stock C (2 pM, 4.54 U/ml): Aliquot 50 μl of Stock B in asterile microfuge tube with 200 μl of intoxication medium at RT (i.e.,1:5 dilution of Stock B).

Materials and Method 3. Plate Coating for Neuronal Cell Culture

Culture plate was coated overnight with either gelatin solution (0.1% in1×PBS), collagen Type IV (0.1 mg/ml), or poly-D-lysine (PDL) (50 μg/ml).Freeze-dried collagen was reconstituted in deionized H₂O to a finalconcentration of 0.1 mg/ml. PDL solution was prepared by dissolving 5 mgpowder in 0.1 M borate buffer, pH 8.5, to the working concentration of50 μg/ml. Culture plate was rinsed twice with 1×DPBS and air-dried inthe tissue culture hood.

Materials and Method 4. Propagation of Cell Lines and Culture Medium

Thirteen neuronal cell lines were collected from 5 different institutes(Table 1). Neuronal cells were purchased from the American TissueCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen (DSMZ), Japanese Collection of Research Bioresources(JCRB), Korean Collection for Type Culture (KCTC), and Korean Cell LineBank (KCLB). They were maintained and propagated in the recommendedmedium. Mycoplasm contamination of all cell lines were monitored usingPCR Mycoplasma Detection Set (Takara Bio Inc. 6601) at every 4 passagesof N2-42F cells or 10 passages of SiMa and hybridoma cells. The PCR testwas performed according to the recommended procedure. In brief, culturesupernatant was collected and incubated for 3-4 days. PCR (50 μl) wasperformed with aliquots (3 μl) of culture supernatant, 1×PCR buffer,dNTP mixture, MCGp F1/R2 primers, and TAKARA EX TAQ™ (Takara Bio RR001).Aliquots (10 μl) of PCR products were resolved on a 1% agarose gel andvisualized by ethidium staining.

TABLE 1 Cell line Source Culture Medium SK-N-SH KCLB MEM, 300 mg/Lglutamine, 30011¹ 25 mM HEPES, 25 mM NaHCO₃, 10% FBS SH-SY5Y KCLB MEM,20 mM HEPES, 22266¹ 25 mM NaHCO₃, 10% FBS IMR-32 KCLB RPMI1640, 300 mg/Lglutamine, 10127¹ 25 mM HEPES, 25 mM NaHCO₃, 10% FBS Neuro-2a KCTC MEM,10% FBS AC28106² SK-N-MC KCTCHC18501² DMEM, 10% FBS N1E-115 ATCCCRL2263³DMEM, 10% FBS NG108-15 ATCC DMEM, 0.1 mM hypoxanthin, HB12317³ 0.4 mMaminopterin, 16 mM thymidine, 10% FBS, 1.5 g/L NaHCO₃ BE(2)-M17 ATCCEMEM + F12, 10% FBS CRL2267³ SiMa DSMZ RPMI1640, ACC164⁴ 2 mM glutamine,10% FBS KP-N-RT-BM-1 JCRB RPMI1640, 10% FBS 1FO50432⁵ KP-N-YNJCRBIFO50431⁵ RPMI1640, 10% FBS NH-6 JCRB 0832⁵ Alpha-MEM, 10% FCS NH-12JCRB 0833⁵ Alpha-MEM, 10% FCS TGW JCRB 0618⁵ EMEM, 10% FBS ¹: KCLB;Korean Cell Line Bank ²: KCTC; Korean Collection for Type Culture ³:ATCC; American Tissue Culture Collection ⁴: DSMZ; Deutsche Sammlung vonMikroorganismen und Zellkulturen ⁵: JCRB; Japanese Collection ofResearch Bioresources FBS; fetal bovine serum, FCS; fetal calf serum

Materials and Method 5: Reagents for Antibodies Development

2-mercaptoethanol (Sigma M3148), 4-iodopheylboronic acid (Sigma 471933),10×TBS (BIO-RAD 170-6435), 10× Tris/Glycine/SDS buffer (Bio-Rad161-0772), 12% Mini-PROTEAN® TGX™ (Bio-Rad 456-1046), acetic acid (Merck100063), alkaline phosphatase (Sigma P0114), AMICON Ultra-15, Ultracel30K (Millipore UFC903024), antibiotic antimycotic solution (AA) (100×)(Sigma A5955), bromophenol blue (Sigma B0126), DMEM (Gibco™ 11995065),DMSO (Sigma D2650), DMSO (Sigma 472301), EZ-link NHS-PEG₄-Biotin (ThermoFisher Scientific 21329), ethylene glycol (Sigma 324558) fetal bovineserum (FBS) (YI Frontier US-FBS-500), glycerol (Affymetrix USB 16374),glycine (Bioshop GLN001), glutaraldehyde solution (Sigma G7651),horseradish peroxidase (Sigma P6782), hydrogen peroxide solution (Sigma216763), LUMINOL (Sigma 123072), magnesium chloride (Sigma M8266), MEMnon-essential amino acid (NEAA) (100×) (GIBCO™ 11140050), methanol(Merck 106009), PCR® STRIP TUBE (Axygen PCR-0208-CP-C), polyvinylidenefluoride (PVDF) membrane (Millipore ISEQ00010), potassium chloride(Sigma P9333), PRECISION PLUS PROTEIN™ dual color standards (Bio-Rad1610374) SDS solution 20% (w/v) (Bio-Rad 161-0418), skim milk (BD DIFCO™232-100), sodium acetate (Sigma W302406), sodium bicarbonate (SigmaS6014), sodium borohydride (Sigma 452882), sodium chloride (Merck106404), sodium (meta)periodate (Sigma S1878), sodium phosphate dibasic(Sigma S7907), sodium phosphate monobasic (Sigma S5011), sodium stannatetrihydrate (Sigma 336262), tetrabutylammonium borohydride (Sigma,230170), T175 (SPL 74175), T75 flask (SPL 70375), Tris (Bioshop TRS001),zinc chloride (Sigma 229997).

Materials and Method 6: Setting of Antibody Manufacturing MethodMaterials and Method 6-1. Generation of Polyclonal and MonoclonalAntibodies Using Synthetic Peptides

Synthetic peptides were conjugated with keyhole limpet hemocyanin (KHL)at either C- or N-terminus, as summarized in FIG. 8. Firstly, tworabbits were immunized with peptide antigens to produce polyclonalserum. After initial injection, the rabbits were boosted periodicallyfor 6 weeks. Rabbit sera were tested for their reactivity andspecificity by Western blot analysis and ELISA. They were then stored at−80° C. before use. Secondly, to generate monoclonal antibody, four micewere injected with peptide antigens. Antibody-producing splenocytes werethen collected and fused with myeloma cells to form hybridomas, followedby three rounds of the single-cell clonal selection, as shown in FIG. 9.In brief, hybridomas were screened initially by ELISA using peptideantigens, then by Western blot analysis with either recombinant SNAP25proteins (GST-SNAP25_(FL) and GST-SNAP25₁₉₇) or total cell lysatesderived from neuronal cells (SiMa), and finally by sandwich ELISA withtotal cell lysates. Antibody-producing hybridomas were expanded andstored at the vapor phase of liquid nitrogen until they were recoveredfor antibody production, as detailed below.

Materials and Method 6-2. Preparation of SNAP25-Affinity Column

Recombinant SNAP25₁₉₇ was concentrated to 10 mg/ml using AMICON Ultra-15by repeated centrifugation at 1,000×g for 10 min at 4° C., whilemeasuring the protein concentration using the Nano spectrophotometer(Drawell Scientific Instrument Co., Ltd, Shanghai). Aliquot (3 ml) ofSNAP25₁₉₇ in AMICO Ultra-15 was mixed with 12 ml of coupling buffer (0.1M HEPES, pH 7.5, 0.1 M NaCl) and re-concentrated by centrifugation at1,000×g. This buffer exchange was repeated 6 times, and SANP25₁₉₇concentrate was added to 1 ml slurry of Affi-Gel 15 (Bio-Rad) indeionized H₂O. After incubation on a shaker incubator for 1 hr at RT,the Affi-Gel 15 was centrifuged at 1,000×g for 10 min at 4° C. ThisSNAP25₁₉₇-conjugated Affi-Gel 15 (SNAP25-AffiGel) was re-suspended in 10ml of 10 mM ethanolamine hydrochloride, pH 8.0 and incubated for 1 hr atRT. After washing with 1×PBS, SNAP25-AffiGel was stored in 1×PBScontaining 0.2% sodium azide before use.

Materials and Method 6-3. Purification of Polyclonal Antibody

Rabbit serum was diluted 10 times with 10 mM Tris-HCl, pH 7.5, andcentrifuged at 10,000×g for 10 min at 4° C. After filtering through 0.45μm bottle top filter (NALGENE™), the clear serum diluent was passedthrough SNAP25-AffiGel three times. SNAP25-AffGel was then washed with20 CV of 10 mM Tris-HCl, pH 7.5, and 0.5 M NaCl, and bound proteins weresequentially eluted with 12 CV of 0.1 M sodium acetate, pH 5.5, 0.1 Mglycine, pH 4.0, and 0.1 M glycine, pH 2.5 and 0.1 M triethylamine, pH11.5. During elution, protein samples were collected in tubes containing0.1 ml of 1 M Tris-HCl, pH 8.0. Protein-containing peak fractions werepooled and concentrated to 1 ml using AMICON Ultra-15. After dialysiswith four changes of 1×PBS and 10% glycerol (0.5 L) at every 90 min,they were analyzed by SDS-PAGE and ELISA.

Materials and Method 6-4. Antibody Conjugation with HorseradishPeroxidase (HRP)

For conjugation of HRP, purified B4 or C16 IgG was concentrated to 10mg/ml using AMICON Ultra-15 through repeated centrifugations at 1,000×gfor ˜15 min per centrifugation at 4° C., while providing with excessivevolume of the conjugation buffer (0.1 M NaHCO₃, pH 9.5, 0.9% NaCl) atthe end of each centrifugation. HRP (5 mg) was solubilized in 1.2 ml ofdeionized H₂O and mixed with 0.3 ml of 0.1 M sodium periodate in 10 mMsodium phosphate, pH 7.0. After incubation for 20 min at RT, the HRPsolution was dialyzed with four changes of 1 mM sodium acetate, pH 4.0,for 6 hr at 4° C. Concentrated antibody (5 mg) and activated HRP (5 mg)were mixed together in a microfuge tube and incubated for 2 hr at RTwith light protection. The conjugation reaction was stopped by theaddition of 0.1 ml of sodium borohydride (4 mg/ml in deionized H₂O). TheHRP-antibody conjugate was dialyzed using Pur-A-Lyzer Maxi 50000 withthree changes of 1×PBS and once with 1×PBS/50% glycerol at an hourlyinterval at 4° C. The HRP-antibody conjugate was stored at 4° C. or −80°C. for a long-term storage before use.

Materials and Method 6-5. Antibody Conjugation with Activated Biotin

A15 IgG (1 mg/ml) was transferred to Pur-A-Lyzer Maxi 20000 and dialyzedwith four changes of a reaction buffer consisting of 0.1 M phosphate, pH7.2, and 0.15 M NaCl at an hourly interval at 4° C. with lightprotection. Activated biotin (10 mM in the reaction buffer) (EZ-LinkNHS-PEG₄-Biotin) was mixed with 50 μg aliquot of A15 IgG to the finalconcentration of 0.1 mM, 0.25 mM, or 0.5 mM. The mixture was adjusted to100 μl with the reaction buffer and incubated for 2 hr at 4° C. withlight protection. After adding 2 μl of 0.1 M glycine, the reactionmixture was dialyzed with three changes of 1×PBS and once with 1×PBS/50%glycerol at an hourly interval at 4° C. and stored in a microfuge tubeat −20° C. before use. The extent of biotinylation of IgG was estimatedusing the Pierce Biotin Quantitation Kit (Thermo Scientific 28005) andthe reactivity and specificity of biotinylated IgG were examined bysandwich ELISA.

Materials and Method 6-6. Crosslinking of AP to Antibodies

Conjugation of alkaline phosphatase (AP) to antibody was initiated byadding glutaraldehyde to 0.25% in a mixture (20

) consisting of reaction buffer (0.1 M sodium phosphate, pH 6.8), 50 μgof AP (2.2 mg/ml) (Sigma-Aldrich P0114-10KU), and 100 μg of purified IgGsuch as monoclonal antibody A15, polyclonal antibody rA15 IgG, andpolyclonal anti-SNAP25 IgG (Sigma-Aldrich S9684). After incubation onice for 1 hr with light protection, the reaction was provided with 1

aliquot of 1 M ethanolamine and incubated for 1 hr at RT with lightprotection. AP-IgG conjugate was dialyzed with three changes of 1×PBSand once with a storage buffer (25 mM Tris-HCl, pH 7.5, 1 mM MgCl₂, 0.1mM ZnCl₂, and 50% glycerol) at an hourly interval at 4° C. Antigenbinding specificity and AP activity of AP-IgG conjugate was examined bydirect ELISA, described below.

Materials and Method 6-7. Measurement of K_(D) by OCTET RED96

For kinetics analysis of monoclonal antibody, the Bio-LayerInterferometry (BLI) assay was performed at 30° C. using FORTÉBIO® OctetRed96 instrument, following the procedure recommended by themanufacturer. In brief, recombinant GST-SNAP25_(FL) or SNAP25₁₉₇ wasdiluted to 125 or 250 nM in 1× kinetics buffer (1×KB)/1×PBS, and theanalyte, i.e. purified IgG, was serially diluted to 3.9, 7.8, 15.6,31.25, 62.5, 125, 250 nM in 1× kinetics buffer. After equilibration in1× kinetics buffer for 1 min, anti-GST probes (FORTÉBIO 18-5096) wereloaded with GST-SANP25_(FL) or GST-SNAP25₁₉₇ for 30 min, followed bydipping in 1× kinetics buffer for 10 min. Subsequent to association anddissociation of analyte for 10 min each, kinetics curves were obtained,and K_(D) (equilibrium dissociation constant) was estimated usingFORTÉBIO® Octet analysis software.

As an alternative kinetics analysis, anti-mouse IgG Fc capture (AMC)biosensors were loaded with antibody and subjected toassociation/dissociation with serially diluted GST-SNAP25. In brief,purified IgG was diluted to 100 or 200 nM, whereas GST-SNAP25_(FL) orSNAP25₁₉₇ was serially diluted to 1.56, 3.125, 6.25, 12.5, 25, 50, and100 nM. After equilibration in 1× kinetics buffer for 1 min, AMCbiosensors were loaded with IgGs for 10 min, followed by dipping in 1×kinetics buffer for 10 min. Association and dissociation of analyte werecarried out for 10 min each, and K_(D) was estimated as described above.

Materials and Method 6-8. Direct ELISA

Immunoplate (Thermo Fisher Scientific A71125) were coated for 2 hr at37° C. with 1 μg of GST-SNAP25_(FL) or GST-SNAP25₁₉₇ in 0.1 M carbonatebuffer, pH 9.5. After washing with 1×PBS, immunoplate was incubated with300 μl of a blocking buffer (5% nonfat dried milk in 1×PBS) for 15 minat RT. After washing three times with 1×PBST (1×PBS/0.05% Tween-20), themicroplate was provided with 100 μl of hybridoma culture supernatant(1:20-10,000 dilution) or ascites fluid (1:1,000-312,500 dilution) andincubated for 1 hr at RT. The microplate was washed three times with1×PBST, to which aliquots (100 μl per well) of goat anti-mouse IgG-HRPconjugate (1:1,000 dilution) (Ab Frontier LF-SA8001) were added. Afterincubation for 1 hr at RT, the microplate was washed three times with1×PBST and HRP reaction was carried out with 50 μL of 1-STEP™ UltraTMB-ELISA (Thermo Fisher Scientific 34028) at RT for 3-25 min. HRPreaction was stopped by the addition of 50 μL of 1 M H₂SO₄ and the ELISAsignal was estimated at 450 nm using BIO-TEK SynergyNeo2.

Materials and Method 6-9. Western Blot Analysis

Recombinant GST-SNAP25 (20 ng-1 μg per well) or total cell lysate ofneuro-2a cells (15 μg per well) were resolved together with Precisionprotein standards (3 μl per well) by 10% or 12% SDS-PAGE. After 5min-soaking in a transfer buffer consisting of 48 mM Tris, 38.9 mMglycine, 20% methanol, 0.05% SDS, proteins were transferred to PVDFmembrane using TRANS-BLOT® Semi-Dry (Bio-Rad 170-3940) for 45 min at 25V PVDF membrane was briefly rinsed with 1×TBST (1×TBS/0.05% TWEEN 20)and incubated with a blocking buffer (5% nonfat dried milk in 1×TBST)for 15 min at RT. Subsequently, PVDF membrane was incubated with eitherhybridoma culture supernatant (1:100 dilution in blocking solution) for45 min at RT. Polyclonal anti-SNAP25 IgG (Sigma 59684) (1:8,000dilution) and anti-SNAP25₁₉₇ IgG (R&D MC6050) (1:100 dilution) were usedas positive controls. After washing with 1×TBST three times for 15 min,PVDF membrane was incubated with either goat anti-rabbit IgG-HRPconjugate (1:10,000 dilution) or goat anti-mouse IgG-HRP conjugate(1:10,000 dilution) for 45 min at RT. After washing 3 times with 1×TBST,recombinant GST-SNAP25 or endogenous SNAP25 was detected and quantifiedusing ECL solution (see below) and Bio-Rad CHEMIDOC™ MP Imaging system(Bio-Rad Universal hood III).

EXAMPLES Example 1: Screening of BoNT/A-Sensitive Neuronal Cell Example1-1. Comparative Analysis of Neuronal Cell Lines for their Sensitivityto BoNT/A

Neuronal cells were seeded at 2×10⁵ cells/well in a 24-well plate and 24hr later, they were treated with 2 nM BoNT/A in medium indicated inTable 1 for 3 days. Total cell lysates (TCLs) were prepared using1×RIPA, and aliquots (3.5 μg) were subjected to 12% SDS-PAGE.

SNAP25_(FL) and SNAP25₁₉₇ were analyzed by Western blot using rabbitpolyclonal IgG (Sigma S9684), specific for SNAP25, as described inMaterials and Methods. ECL solution was formulated and optimized byAbBio Inc. Working ECL solution was prepared by mixing Sol A and Sol Bin 1:1 ratio before use. Sol A and Sol B is comprised as Table 2. Andsensitivity of cell lines to BoNT/A (2 nM) were represented in Table 3.

TABLE 2 ECL solution Composition SolA 0.1 M Tris-HCl, pH 8.8, 2.5 mMLUMINOL in DMSO, 4 mM 4-iodopheylboronic acid, 0.2 mM tetrabutylammoniumborohydride, 2% ethylene glycol, and 0.02% TRITON X-100. SolB 0.1 MTris-HCl, pH 8.8, 10.6 mM hydrogen peroxide, and 0.012% sodium stannate

TABLE 3 Cell line Sensitivity to BoNT/A (2 nM) SK-N-SH No SH-SY5Y NoIMR-32 No Neuro-2a Yes SK-N-MC No N1E-115 No NG108-15 No BE(2)-M17 NoSiMa Yes KP-N-RT-BM-1 Yes KP-N-YN No NH-6 No NH-12 No TGW No

As summarized in Table 3, except for SiMa cell, only neuro-2a andKP-N-RT-BM1 (K-BM1) cells yielded detectable levels of SNAP25 cleavage.

Example 1-2. Screening of BoNT/A-Sensitive Neuronal Cell

Neuro-2a, SiMa and KP-N-RT-BM-1 cells were separately cultured on a24-well plate under the same conditions as materials and method, and thecleavage phenomenon of SNAP25 protein was analyzed by Western blotanalysis The results are shown in FIG. 1. The extents of SNAP25 cleavagewere estimated about 33%, 66%, and 29% for neuro-2a, SiMa, and K-BM1cells, respectively.

Neuro-2a was further used for clonal selection over K-BM1 for thefollowing reason. Firstly, the in vivo potency of BoNT/A, which isconventionally measured by mouse lethality (i.e. mouse LD₅₀), could bebetter recapitulated with mouse cell line. In fact, neuro-2a is a mousecell line, but K-BM1 is a human cell line. Secondly, K-BM1 tends to growat a much slower rate than neuro-2a (data not shown). It should be notedthat the population doubling time of SiMa cells, a human neuroblastomacell line, is reported to be 34 to 100 hrs (DSMZ ACC-164). Thirdly,neuro-2a cells appear to be a mixed population of several cell typesunder the microscope. Thus, it is likely that BoNT/A-sensitive cellsexist in the heterogeneous neuro-2a cell population.

Example 2: Clone Selection for BoNT/A-Sensitive Neuronal Cell

Among the Neuro-2a cell populations selected in Example 1, clones highlysensitive to BoNT/A were selected.

Example 2-1. Clonal Culture of Neuro-2a Cells

Neuro-2a cells were propagated in 1MEM supplemented with 10% fetalbovine serum (FBS), 1× non-essential amino acids (1×NEAA), 1× sodiumpyruvate, 1×GLUTAMAX™, and 1× antibiotic antimycotic (1×AA). When cellsreached ˜80-90% confluence, they were treated with 1× TrypLE (GIBCO™12604021) to make a single cell suspension. After determining the viablecell number using the hemocytometer and trypan blue, cells were dilutedto the density of 10 cells per ml of medium. Then, 100 μl aliquots (˜onecell equivalent) were added to 7×96-well culture plate. Colony growthwas periodically monitored by microscopic examination. At ˜60%confluence, cells were transferred to a 24-well culture plate. Later,they were equally divided, and one half was stored in the liquidnitrogen tank, and the rest was tested for the sensitivity to BoNT/Aintoxication.

Neuro-2a clonal cells were sub-cultured from the 24-well plate to96-well microplate. On the following day, culture medium was replacedwith PRMI1640 supplemented with 2 mM L-alanyl-L-glutamine, 1×B27, 1×N2,1×NEAA (differentiation medium) for 2 days to induce neuronaldifferentiation. On the 4^(th) day, GT1b was added to the finalconcentration of 25 μg/ml (1× intoxication medium). After incubation for24 hr, the culture medium was replaced with the intoxication mediumcontaining BoNT/A (0.1 nM), and cells were incubated for additional 2days. After removing medium by aspiration, cells were treated with 1×SDSsample buffer, and aliquots were examined for the extent of SNAP25cleavage by 12% SDS-PAGE and Western blot analysis. This clone selectionprocedure was repeated three times, and the results confirmed in eachclone selection procedure are shown in FIG. 2. Following the procedureoptimized for SiMa cells by Allergan with minor modifications asdescribed in Materials and Methods (PLoS One. 2012; 7(11):e49516), 142clonal cells, plated from 24-well plates to 96-well culture plates, weretested for the sensitivity to BoNT/A. As shown in FIG. 2, a total of 19positive clones were identified that exhibited noticeably highersensitivities to BoNT/A than parental neuro-2a. Of positive clonessubjected to multiple rounds of a single-cell clonal selection, onlyclone 42 persistently displayed higher BoNT/A sensitivity than parentalneuro-2a. After three rounds of a single-cell clonal selection, clone 42was re-named as N2-42F following the nomenclature recommended for celllines (Cell Stem Cell. 2011 Jun. 3; 8(6):607-8; Bioinformatics. 2008Dec. 1; 24(23):2760-6).

Example 2-2. Recovery, Proliferation, and Storage of Freezed Cells

Recovery of freezed cells was performed as follows.(1) Rapidly thaw (<2 minute) the cell stock vial, retrieved from aliquid nitrogen freezer, by gentle agitation in a 37° C. water bath.(2) Once thawed, decontaminate the cell stock vial by spraying with 70%ethanol.(3) Unscrew the top of the vial in a laminar flow tissue culture hood,and transfer the content to a sterile 15-ml conical tube containing 9 mlof pre-warmed complete medium.(4) After gentle centrifugation (125×g for 10 min), remove thesupernatant by aspiration and re-suspend the cells in 2 ml of thecomplete medium.(5) Pipet gently to loosen the pellet and break apart clumps, andtransfer the cell suspension to T75 flask containing 25 ml of thecomplete medium.(6) Incubate the flask at 37° C. in a CO₂ incubator.Cell culture was performed as follows.(1) When cells become ˜90% confluent, remove the culture medium byaspiration and rinse the culture flask once with 1×DPBS.(2) Add 1 ml of 0.25% trypsin-EDTA and incubate the flask for 10 min at37° C.(3) Add 9 ml of the complete medium, and break apart cell clumps byrepeated pipetting.(4) Transfer the cell suspension to a 15 ml-conical tube, and centrifugeit at 800×g for 5 min.(5) Discard the supernatant by aspiration and re-suspend the cell pelletwith 10 ml of the complete medium.(6) Determine the density of viable cells using the hemocytometer.(7) Transfer 3 ml aliquots of the cell suspension to fresh T75 flasks.(8) Incubate for 3 days until cells reach ˜90% confluency.Cell freezing for establish cell bank was performed as follows.(1) Subculture ˜90% confluent cells in two T75 flasks to eight T75flasks.(2) When cells reach a late growth phase (˜2.0×107 cells/flask), removethe culture medium and prepare a single-cell suspension throughtreatment with 0.25% trypsin-EDTA.(3) Re-suspend the cell pellet at a density of 5×106 cells/ml with afreezing medium.(4) Aliquot 1 ml of cell suspension into cryogenic stock vials.(5) Place tubes in the isopropanol-filled freezing container (be sure totighten the tube cap).(6) Transfer the freezing container to −80° C. overnight.(7) On the following day, transfer frozen cells immediately to theliquid nitrogen vapor phase storage.

Example 3: Identification of N2-42F Characteristics and CultureEnvironment Example 3-1. Confirmation of Doubling Time of N2-42F

The cleavage times of Neuro-2a cells and N2-42F, which were confirmed tobe capable of detecting truncated forms of the SNAP25 protein, weredetermined.

N2-42F and neuro-2a cells were seeded at 1.5×10⁵ cells per well in a6-well culture plate. On a daily basis for 6 days, total viable cellswere counted using the hemocytometer and trypan blue staining. Doublingtime was calculated using viable cell numbers obtained during theexponential growth phase as follows Table 4. And the results are shownin Table 5 and FIG. 3. Tis incubation time, Xb and Xe are the cellnumber at the beginning and at the end of incubation time in Table 4,respectively.

TABLE 4 Doubling time = T X ln2/ln(Xe/Xb)

TABLE 5 Cell line Doubling time (hr) Neuro-2a 24 ± 4.7 N2-42F 24 ± 2.9

As shown in Table 5 and FIG. 3, the cleavage time was 24±4.7 hours forNeuro-2a and 24±2.9 hours for N2-42F.

Example 3-2. Confirmation of Morphology of N2-42F

Allergan reported the isolation of BoNT/A-sensitive clone (H1) from SiMacells (PLoS One. 2012; 7(11):e49516). Thus, it is expected that likeneuro-2a, SiMa cells also represent a heterogeneous cell population ofmixed cell types but their sub-clones such as H1 and N2-42F arehomogeneous cell types. This was confirmed by microscopic examination ofneuro-2a, N2-42F, and SiMa cells. As shown in FIG. 4, both neuro-2a andSiMa exhibit heterogeneous morphologies under the microscope but N2-42Fcells look highly homogeneous.

From the above results, it can be seen that N2-42F corresponds to ahomogeneous cell type as a single clone among various clonesconstituting Neuro-2a.

Example 3-3. Confirmation of Culture Conditions of N2-42F

In efforts to optimize the BoNT/A intoxication condition, N2-42F cellswere grown in culture plates coated with different matrices. Unlikeparental neuro-2a cells that are routinely propagated in non-coatedculture plates, N2-42F cells exhibit a clear preference for cultureplates coated with poly-D-lysine (PDL) (FIG. 5). Neither non-coatedplates nor the ones coated with collagen type IV or gelatin supportedefficient growth of N2-42F cells. On those plates, N2-42F lookedunhealthy, forming clumps. Also, they were loosely attached to theplate. By contrast, N2-42F cells were grown on PDL-coated plates, firmlyattached yet evenly distributed. Thus, unless otherwise indicated,PDL-coated culture plates were exclusively used for N2-42F cells in thepresent research.

Example 4: Confirmation of the Susceptibility of N2-42F to BotulinumNeurotoxins

Using SiMa cells as control, the BoNT/A sensitivity of N2-42F cells wasthoroughly examined in triplicate sets of 96-well plate culture providedwith 1× intoxication medium containing varying concentrations of BoNT/A.After incubation for 4 days, cells were treated with 50 μl of 1×SDSsample buffer, and aliquots (12 μl) were subjected to Western blotanalysis using polyclonal anti-SNAP25 IgGs (Sigma S9684), as describedin Materials and Methods. Endogenous SNAP25 cleavage was insignificantlydetected in both SiMa and N2-42F cells treated with 0.93 pM or lowerconcentrations of BoNT/A. Treatment with 2.78-25 pM BoNT/A led to 25-72%cleavage of SNAP25 in N2-42F cells and 33-75% with SiMa cells (FIG. 6a). This result indicates that N2-42F cells are as sensitive as SiMacells.

Including BoNT/A, there are seven serologically different botulinumneurotoxins from BoNT/A to BoNT/G. Similar to BoNT/A, BoNT/B has alsobeen licensed for the pharmaceutical application such as MYOBLOC® orNEUROBLOC®. Thus, it was explored if N2-42F cells can be used in anycell-based potency assays for different serotypes of botulinumneurotoxins. For this purpose, differentiating N2-42F and Neuro-2a cellswere compared for their susceptibility to different neurotoxin complexes(Metabiologics C08RA188-RA194).

As shown in FIG. 6 b, 25 pM of Metabiologics BoNT/A (M-BoNT/A) gave riseto a saturating extent of SNAP25 cleavage in N2-42F. Under the samecondition, about 44% of SNAP25 cleavage was observed in Neuro-2a cells.The higher sensitivity of N2-42F cells to M-BoNT/A is consistent withits sensitivity to BoNT/A, prepared by HUGEL (i.e. Botulax). N2-42Fcells also exhibited significantly a higher sensitivity to M-BoNT/B.Intoxication with 2 nM of M-BoNT/B resulted in near complete cleavage ofVamp2 in N2-42F cells but only about 50% cleavage in Neuro-2a. Though tolesser extents, N2-42F cells exhibited higher sensitivities to M-BoNT/Cand M-BoNT/F. But with M-BoNT/D (5 pM or 200 pM), there was nonoticeable difference in their sensitivity between N2-42F and Neuro-2acells (data not shown). Nor they showed any detectable level ofsensitivity to M-BoNT/E (10-400 pM) or BoNT/G (50-2000 pM). Our resultsindicate that N2-42F cells can be used as host in the cell-based potencyassay for BoNT/A, BoNT/B, BoNT/C, and BoNT/F.

Example 5: Confirmation of Stability of N2-42F

Passage stability could a key feature of neuronal cells to be used ashost in any cell-based assay platforms. N2-42F cells were continuouslypropagated for multiple passages. The cells in early passages were usedto make the master cell bank as described in Materials and Methods. Andat every 5 passages, cells were also stored in the liquid nitrogen tanksfor the passage stability. In brief, N2-42F cells stored at passage 5(P5) and 15 (P15) were restored from the liquid nitrogen tank and at˜90% confluence, they were compared in the BoNT/A sensitivity using the1× intoxication medium containing 0.1 nM BoNT/A following the proceduredescribed in FIG. 6. Experiment was performed in a triplicate set usingSiMa cells as control. As shown in FIG. 7, 63-68% of SNAP25 cleavage wasmeasured in N2-42 cells at P5 and 63-71% at P15. SiMa cells exhibited70-77% cleavage.

This result indicates that similar to H1 clone of SiMa cells identifiedby Allergan, the BoNT/A sensitivity is a stably inheriting property ofN2-42F cells. which makes them a suitable host, the second of its kindnext to the H1 clone of SiMa cells, in a cell-based assay platform.

Example 6: Determination of BoNT/a Potency Based on N2-42F Example 6-1.Experimental Method

96-well plates were coated in the manner described in Materials andmethods. While air-drying the 96-well culture plate, prepare a total of7 ml of N2-42F cell suspension in the density of 5.5×10⁵ cells/ml. About90% confluent N2-42F cells in one T75 flask would be sufficient forthree 96-well culture plates. Transfer the cell suspension to a sterilebuffer reservoir, and dispense aliquots (100 μl) of the cell suspensioninto each well using a multichannel pipette, and incubate the 96-wellculture plate in a CO2 incubator. Outer wells of a 96-well plate shouldbe filled with aliquots (100 μl) of 1×AA solution to avoid the dreadededge effects.

On the day after the cells were dispensed, all cell culture medium wasremoved from the 96-well plate using a multi-channel pipette, and 100 μlof RPMI 1640 was added to rinse. The BoNT/A intoxication medium was thentreated to each 96-well plate and incubated for 4 days at 37° C., 5%CO₂. In addition, the capture antibody, B4 IgG, was prepared in anamount of 7 ml, which was then divided into 50

in ELISA plates and stored at 4° C. overnight.

For measurement, the BoNT/A intoxication medium was removed from the96-well plate using a multi-channel pipette and each well was treatedwith 60 μl of a lysis buffer (pH 7.5, 20 mM HEPES, 1% TRITON-200 mMNaCl, 1 mM EGTA, and 5 mM EDTA, added with proteolysis inhibitorimmediately before use), and incubated 4° C. for 20 minutes with shakingat a speed of 500 rpm. Thereafter, the dissolution buffer contained ineach well was obtained, and centrifuged 4,000 rpm for 20 minutes at 4°C.

The ELISA plate was washed 3 times with the washing buffer, added with300 μl aliquots of the blocking buffer to each well, incubated for 15min at RT, and washed twice with the washing buffer after remove theblocking buffer.

50 μl aliquots of TCL was transferred from the 96-well culture plate tothe ELISA plate coated with the capture antibody (B4), and the ELISAplate was incubated for 4 hr at 4° C. on a microplate shaker at 200 rpm.Finally, the plate washed 3 times with the wash buffer.

For detection of SNAP25 and SNAP25₁₉₇, detection antibodies were addedto ELISA plate (50 μl per well), and the plate was incubated for 1 hr atRT on a Thermo shaker incubator (200 rpm). And the plate was rinsedthree times with the wash buffer, 50 μl aliquots of 1-Step™ UltraTMB-ELISA was added to the plate, the HRP reaction was terminated byadding 2 M sulfuric acid (50 μl/well) after 5 min. The HRP reaction wasmeasured at 450 nm. The value at A₄₅₀ may represent the relative amountof SNAP25₁₉₇.

Example 6-2. Preparation of Standard Curve and Determination of theBoNT/a Potency

Standard curve and BoNT/A Potency were tested in the following manner.

Calculate the average A450 value of control wells where sandwich ELISAwas carried out with no BoNT/A treatment (i.e. 0 pM). Subtract theaverage control A450 value from test A450 values, and calculate thenormalized average test A450 value. And then, plot the normalizedaverage A450 values on Y axis against BoNT/A potency on X axis usingPrism 5.0 (GraphPad Software, La Jolla, Calif.). Analyze the plot bysuccessively selecting “analyze”, “nonlinear regression (curve fit)”,and “sigmoidal dose-response”, which will yield a EC50 value. Preparethe standard curve using the normalized A450 values of test wellstreated with 0.1-0.93 pM BoNT/A Standard Reference (see Appendix 2,Section D). And use the standard curve equation with R2 value of 0.95 orhigher to determine the BoNT/A potency of test samples.

Example 7: Generation of Monoclonal Antibodies Specific for SNAP25

Allergan used a 13-amino acid (AA) residue peptide N-CDSNKTRIDEANQ-C(SEQID NO: 91) to raise antibody. The peptide was designed to be identicalto the C-terminal end of SNAP25₁₉₇ generated upon BoNT/A digestion.Since SNAP25_(FL) also has the identical amino acid sequence in it, itwould be reasonable to posit that the specificity of monoclonalantibodies recognizing SNAP25₁₉₇ is attributable to as yet unidentifiedfeature of SNAP25₁₉₇ rather than its primary amino acid sequence. SNAP25consists of 206 AA residues (FIG. 8). Through SNARE motifs, it forms astable ternary complex with syntaxin 1A and synaptobrevin 2 (VAMP2). Theless stable and non-functional ternary complex is formed with SNAP25₁₉₇,whereas the ternary complex fails to form at all with SNAP₁₈₀ (PeerJ.2015 Jun. 30; 3:e1065). Their finding that the C-terminal 9 AAs ofSNAP25 is essential for the in vivo function and formation of a stableternary complex suggests that the cleavage at AA position 197 by BoNT/Ainduces as yet unidentified structural alteration, particularly at theC-terminus and the second SNARE domain. Thus, the postulated structuraldifference between SNAP25_(FL) and SNAP25₁₉₇ makes it possible toproduce monoclonal antibodies specific for SNAP25₁₉₇.

A total of 10 peptide antigens were designed to meet the followingcriteria (Table 6). First, alpha helical regions exhibiting relativelylower antigenicity were excluded. Second, peptide sequences arenon-redundant and unique, the properties of which are essential toreduce cross reactivity of antibodies.

TABLE 6 Immunogens/AA  SEQ Position in SNAP25 ID NOs AA SequenceN peptide   1-13 SEQ ID NO: 1 KLH-C-MAEDADMRNELEE   1-13 SEQ ID NO: 2MAEDADMRNELEE-C-KLH  19-38 SEQ ID NO: 3 KLH-C- DQLADESLESTRRMLQLVEE 51-70 SEQ ID NO: 4 KLH-C- DEQGEQLERIEEGMDQINKD M peptide 122-136SEQ ID NO: 5 KLH-C-DEREQMAISGGFIRR C peptide 170-184 SEQ ID NO: 6KLH-C-EIDTQNRQIDRIMEK 180-194 SEQ ID NO: 7 KLH-C-RIMEKADSNKTRIDE 180-197SEQ ID NO: 8 KLH-C-RIMEKADSNKTRIDEANQ 186-197 SEQ ID NO: 9KLH-C-DSNKTRIDEANQ 189-201 SEQ ID NO: 10 KLH-C-KTRIDEANQPATK

Of a total of 10 peptides described in Table 6, M and N peptides wereused to generate monoclonal antibodies capable of detecting bothSNAP25_(FL) and SNAP25₁₉₇. With C peptides, it was anticipated to obtainthree different monoclonal antibodies with the binding specificitytoward (1) SNAP25_(FL), (2) SNAP25₁₉₇, and (3) both SNAP25_(FL) andSNAP25₁₉₇. Throughout our research, two commercially availableantibodies were used as control in ELISA and Western blot analysis. Theyinclude rabbit polyclonal anti-SNAP25 IgGs (Sigma-Aldrich) and MC6050(R&D). The former recognizes both SNAP25_(FL) and SNAP25₁₉₇, but thelatter is a monoclonal antibody specific for SNAP25₁₉₇.

Individual synthetic peptide was injected to two rabbits to raisepolyclonal serum and 4 mice to raise monoclonal antibody, as describedin Materials and Methods. In particular, the procedure to establishhybridoma cells producing monoclonal antibody was summarized in FIG. 9.In brief, four mice were immunized per peptide antigen to induceantibody production. After ELISA screening of mouse serum for antibodyproduction, splenocytes were isolated from ELISA-positive mice and fusedwith SP2 myeloma cells. Subsequently hybridoma cells were seeded at asingle cell density 96-well plate per peptide antigen. As summarized inFIG. 9, initial screening to obtain positive clones was performedemploying direct ELISA using peptide antigen, which was followed by amore stringent screening utilizing recombinant SNAP25 and total celllysate.

A representative result of initial hybridoma screening is shown in FIG.10. Culture supernatants were collected from hybridoma cells (clone 4,B4) grown in 7×96-well plate and tested for their reactivity to peptideantigen by direct ELISA. About 25% clones yielded positive ELISA signals(panels A-G), and 24 relatively strong positives were selected andtested for their reactivity to endogenous SNAP25 by sandwich ELISA usingtotal cell lysate (panel H). Ten clones selected for multiple rounds ofsingle-cell clonal selections.

For the single-cell clonal selection, hybridoma clones were seeded at asingle-cell density in one 96-well plate. Four days later, culturesupernatants were tested for the reactivity to recombinant SNAP25 bydirect ELISA, and five clones yielding relatively strong ELISA signalswere selected for further analysis. For example, in case of clone 4,over 80% sub-clones, grown in a 96-well plate, were tested positive indirect ELISA (FIG. 11a ). Twelve sub-clones produced ELISA signals lowerthan negative control, including A8-A10, B11, C10, D4, D8, D9, E5, E11,F10, and G7 (FIG. 11a ). Five sub-clones were also analyzed by sandwichELISA (FIG. 11b ) and Western blot (FIG. 11c ) using total cell lysate(TCL), as described in Materials and Methods. It should be noted thatELISA signals, with sub-clones of clone 4, were in a good agreement withthe Western blot signals. Since TCL underwent denaturation duringWestern blot analysis, this result suggests that monoclonal antibodyproduced by hybridoma clone 4 equally reacts with both non-denatured(i.e. ELISA) and denatured SNAP25.

Single-cell clonal selection repeated two more rounds, as summarized inFIGS. 12 & 13. After the second round of selection, two clones 6 & 8were dropped from further screening because all culture supernatants in96-well plate failed to yield ELISA signals significantly higher thannegative control. Through a series of single-cell clonal selection, atotal of 12 hybridoma clones were obtained that produce monoclonalantibodies specific for SNAP25_(FL), SNAP25₁₉₇, or both.

Example 8: Production and Purification of Monoclonal Antibodies

Hybridoma cells were expanded in T175 flasks and used to make mastercell bank. For production of monoclonal antibody, hybridoma cells wererecovered from the stock vial stored at the vapor phase of liquidnitrogen. When cells reached about 90% confluency, they weresub-cultured at 1:4-10 in multiple T175 flasks. After incubation for 3-4days, cells were collected by centrifugation and re-suspended inserum-free medium at 1.0×10⁶ cells/ml. After incubation for 4 days,culture supernatants were collected and used to purify IgG using proteinG column (HITRAP® Protein G HP column), as described in Materials andMethods. Yield of monoclonal antibody purified using culturesupernatants varied batch to batch of hybridoma culture and also forindividual hybridoma clone. Over a dozen times of purification withdifferent batches of culture supernatant (165-542 ml) yielded about2.45-5.52 mg of C16 IgG (Table 7).

TABLE 7 Monoclonal Volume of cell culture Yield antibody supernatant (

) (mg) C16 IgG₁ 185 2.78 235 4.32 542 2.90 331 2.45 291 2.60 233 5.52225 3.87 253 3.55 190 3.16 165 3.35 269 3.76 283 3.92 277 4.41 299 4.13237 3.43

Purification of other monoclonal antibodies is summarized in Table 8.

TABLE 8 Polyclonal Volume of cell culture Yield antibody supernatant (

) (mg) A15 IgG₁ 403 6.37 210 2.22 202 4.20 217 3.90 171 3.45 226 3.16233 5.52 205 4.04 200 3.63 177 5.11 B23 IgG₁ 44.5 0.17 186 1.14 B20 IgG₁45 0.58 B16 IgG₁ 44.5 0.39 B4 IgG₁ 42 1.34 292 4.32 282 6.42 285 5.45275 3.40 C7 IgG_(2a) 152 5.18 161 3.30 F14 IgG₁ 263 2.56

On average, about 1-2 mg of IgG was obtained from 100 ml of culturesupernatant. Though data not shown, the purity of IgGs and their antigenbinding specificity were validated by SDS-PAGE and ELISA.

Example 9: Purification of Polyclonal Antibody

Polyclonal antibody was purified from rabbit serum using SNAP25-AffiGel,as described in Materials and Methods. In brief, rabbit serum wasdiluted 10 times with 10 mM Tris-HCl, pH 7.5, and cleared bycentrifugation at 10,000×g for 10 min and filtering through 0.45 μmfilter. The clear serum diluent was repeatedly loaded onto aSNAP25-AffiGel three times, and bound proteins were sequentially elutedwith 0.1 M sodium acetate, pH 5.5, 0.1 M glycine, pH 4.0, 0.1 M glycine,pH 2.5, and 0.1 M triethylamine, pH 11.5. Protein fractions werecollected in tubes containing 0.1 ml of 1 M Tris-HCl, pH 8.0, pooled andconcentrated to 1 ml using AMICON Ultra-15. After dialysis with fourchanges of 1×PBS and 10% glycerol at every 90 min, they were analyzed bySDS-PAGE and ELISA.

Table 9 summarizes the relative distribution of serum proteins in theSNAP25-AffiGel fractions obtained with 4 different batches of rA15 sera.

TABLE 9 Rabbit Yield (μg) serum (

) pH 5.5 pH 4.0 pH 2.5 30 10 85 350 30 1102 525 1160 30 936 504 990 10064 337 945

Despite that each serum sample exhibited different protein distributionpatterns in SNAP25-AffiGel fractions (Table 9), IgG was detected as amajor constituent of all fractions examined by 10% SDS-PAGE (FIG. 14a ).When tested in sandwich ELISA, however, the reactivity to endogenousSNAP25 was detected only with pH 4.0 fraction. A high signal tobackground ratio (>25) (FIG. 14b ) indicates that IgG in pH 4.0 fractionis highly specific for endogenous SNAP25. Since similar results wereobtained with other rabbit sera, pH 4.0 fraction was exclusively usedthroughout our study employing polyclonal serum unless otherwiseindicated.

Example 10: Sequence Analysis of Monoclonal Antibodies

Total RNA, extracted from hybridoma cells, were reversed transcribed tocDNA using either an oligo-dT anti-sense primer or a gene-specific(murine IgG1 CH and kappa CL) anti-sense primer. Specific murineconstant domain primers were used to amplify the cDNA by PCR todetermine the isotype of the antibody. Degenerate V_(H) and V_(L),primers were used to amplify the variable domains from the cDNA. For 5′RACE, a homopolymeric [dC] tail was added to the 3′ end of the cDNA. Theheavy and light chains were then amplified with an oligo [dG] senseprimer and a gene specific (CH/KC) anti-sense primer. The PCR productswere cloned into a blunt or TA vector for sequencing. The sequencingresults were aligned to V_(H) and V_(L) chains to determine consensussequences.

CDR sequences are summarized in three different groups of IgGs accordingto their antigenic specificity: (1) SNAP25_(FL)-specific IgGs (Table10), (2) SNAP25₁₉₇-specific IgGs (Table 11), and (3) Bi-specific IgGs,reacting with both SNAP25_(FL) and SNAP25₁₉₇, (Table 12).

TABLE 10 Identified CDR SEQ ID NOs Sequence In V_(H) CDR1 SEQ ID NO: 11GYSITSGYY D2 SEQ ID NO: 12 GYTFTDYN D6 SEQ ID NO: 13 GYTFTNYG E6V_(H) CDR2 SEQ ID NO: 14 IRYDGSN D2 SEQ ID NO: 15 IYPYNGDT D6SEQ ID NO: 16 INTYTGEP E6 V_(H) CDR3 SEQ ID NO: 17 ARDRDSSYYFDY D2SEQ ID NO: 18 VRSGDY D6 SEQ ID NO: 19 ARGYYDY E6 V_(L) CDR1SEQ ID NO: 20 DHINNW D2 SEQ ID NO: 21 QSLLDSNGKTY D6 SEQ ID NO: 22QSLLDSDGKTY E6 V_(L) CDR2 SEQ ID NO: 23 DTT D2 SEQ ID NO: 24 LVS D6, E6V_(L) CDR3 SEQ ID NO: 25 QQYWSAPPT D2 SEQ ID NO: 26 WQGTLFPYT D6SEQ ID NO: 27 WQGTHFPRT E6

TABLE 11 CDR SEQ ID NOs Sequence Identified In V_(H) CDR1 SEQ ID NO: 28GYSITSDYA C4 SEQ ID NO: 29 GFTFNTNA C7, C14 SEQ ID NO: 30 GYTFTNYT C16SEQ ID NO: 31 GYTFNTYA C24 SEQ ID NO: 32 GFTFSNYG D3 SEQ ID NO: 33GFTFNTYA C15 V_(H) CDR2 SEQ ID NO: 34 ISYSVGT C4 SEQ ID NO: 35IRSKSNNYAT C7, C15 SEQ ID NO: 36 IRSKSDNYAT C14 SEQ ID NO: 37 INPSSDYTC16 SEQ ID NO: 38 IRSKSNNYTT C24 SEQ ID NO: 39 INSNGGTT D3 V_(H) CDR3SEQ ID NO: 40 ARKGEYGFAY C4 SEQ ID NO: 41 VYGRSYGGLSY C7 SEQ ID NO: 42VYGRSYGGLGY C14 SEQ ID NO: 43 VRQVTTAVGGFAY C15 SEQ ID NO: 44ARRIFYNGRTYAAMDY C16 SEQ ID NO: 45 VGQILYYYVGSPAWFAY C24 SEQ ID NO: 46ARDRDAMDY D3 V_(L) CDR1 SEQ ID NO: 47 KSVSTSGYSY C4, C14, C24SEQ ID NO: 48 KSVSSSGYSY C7, C15, C16 SEQ ID NO: 49 QSIVNSHGNTY D3V_(L) CDR2 SEQ ID NO: 50 LAS C4, C7, C14, C15, C16, C24 SEQ ID NO: 51KVS D3 V_(L) CDR3 SEQ ID NO: 52 QHSRELPLT C4, C7, C14, C15, C16SEQ ID NO: 53 QHSRELPWT C24 SEQ ID NO: 54 FQGSHVPWT D3

TABLE 12 Identified CDR SEQ ID NOs Sequence In V_(H) CDR1 SEQ ID NO: 55GFTFSNYG B4 SEQ ID NO: 56 GINIKDYY B23 V_(H) CDR2 SEQ ID NO: 57 ISSGGSYTB4 SEQ ID NO: 58 IDPGNGDA B23 V_(H) CDR3 SEQ ID NO: 59 ARREGGGNPYFDY B4SEQ ID NO: 60 NEIAY B23 V_(L) CDR1 SEQ ID NOs: 61 QSLVHSNGNTY B4SEQ ID NO 62 QSLLDSDGKTY B23 V_(L) CDR2 SEQ ID NO: 63 KVS B4SEQ ID NO: 64 LVS B23 V_(L) CDR3 SEQ ID NO: 65 SQNTLVPWT B4SEQ ID NO: 66 WQGTRFPFT B23

The V_(H) and V_(L) domain sequences of the antibodies produced in thepresent invention are summarized in Table 13.

TABLE 13 Antibody SEQ ID NOs Sequence B4 V_(H) SEQ ID NO: 67EVKLVESGGGLVKPGGSLKLSCAASGFTFSNYGMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARREGGGNPYFDY WGQGTTLTVSS V_(L) SEQ ID NO: 68DVLMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQNTLVPWTFGGGTKLEIK B23 V_(H) SEQ ID NO: 69EVQLQQSGAELVRPGASVKLSCTASGINIKDYYMHW MKQRPEQDLEWIGWIDPGNGDAEYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNEIAYWGQGTLV TVSA VL SEQ ID NO: 70DIVMTQSPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPFTFGSGTKLEIK C4 V_(H) SEQ ID NO: 71DVKLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYISYSVGTRYNPSLKSRISITRDTSKNQFFLLLKSVTNEDTATYFCARKGEYGFAYWGQGT LVTVSA V_(L) SEQ ID NO: 72DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C7 V_(H) SEQ ID NO: 73QVQLVETGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSLLYLQMNNLKTEDTAMYYCVYGRSYGGLS YWGQGTLVTVSA V_(L) SEQ ID NO: 74DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C14 V_(H) SEQ ID NO: 75EVKLVESGGGLVQPKGSLKLSCAASGFTFNTNAMNWVRQAPGKGLEWVARIRSKSDNYATYYADSVKDRFTISRDDSPSMLYLQMNNLKTEDTAMYYCVYGRSYGGLG YWGQGTLVTVSA V_(L) SEQ ID NO: 76DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYVHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C15 V_(H) SEQ ID NO: 77EVKLVESGGGLVQPKGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSQSMLYLQMNNLKTEDTAMYYCVRQVTTAVGG FAYWGQGTLVTVSE V_(L) SEQ ID NO: 78DIVMTQSPASLAVSLGQRTTISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELR C16 V_(H) SEQ ID NO: 79EVQLQQSGAELARPGASVQMSCKAFGYTFTNYTMHWVRQRPGQGLEWIGFINPSSDYTNYNQKFKDKATLSADKSSSTAYMQLSSLTSEDSAVYYCARRIFYNGRTYAA MDYWGQGTSVTVSS _(VL) SEQ ID NO: 80DIVMTQSPASLAVSLGQRATISCRASKSVSSSGYSYMHWYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIFIPVEEEDAATYYCQHSRELPLTFGAGTKLELK C24 V_(H) SEQ ID NO: 81EVKLVESGGGLVQPKGSLKLSCAASGYTFNTYAMNWVRQAPGKGLEWVARIRSKSNNYTTYYADSVKDRFTISRDDSQSMLYLQINNLKTEDTAMYYCVGQILYYYVGS PAWFAYWGQGTLVTVSA V_(L)SEQ ID NO: 82 DIVMTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWYQQKPGQPPKLLIFLASNLESGVPARFSGSGSGTDFTLNIFIPVEEEDAATYYCQHSRELPWTFGGGTKLEIK D2 V_(H) SEQ ID NO: 83DVKLQESGPGLVKPSQSLSLTCSVTGYSITSGYYWNWIRQFPGNKLEWMGYIRYDGSNNYNPSLKNRISITRDTSKNQFFLKLNSVTTEDTASYYCARDRDSSYYFDYWGQ GTALTVSS V_(L) SEQ ID NO: 84DIVMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISDTTSLETGVPSRFSGSGSGKDYTLSIT SLQTEDVATYYCQQYWSAPPTFGGGTKLEIKD3 V_(H) SEQ ID NO: 85 EVQLEESGGGLVQPGGSLKLSCAASGFTFSNYGMSWVRQTPDKRLELVATINSNGGTTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDSAMYYCARDRDAMDYWGQ GTSVTVSS V_(L) SEQ ID NO: 86DVLMTQTPLSLPVSLGDQASISCRSSQSIVNSHGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIK D6 V_(H) SEQ ID NO: 87EVQLQQSGPELVKPGASVKISCKASGYTFTDYNMHWVKQSHGKSLEWIGYIYPYNGDTGYNQKFKSKATLTVDNSSSTAYMELRSLTSEDSAVYYCVRSGDYWGQGTT LTVSS V_(L) SEQ ID NO: 88DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSNGKTYLNWLLQRPGQSPSRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTLFPYTFGGGTKLEIK E6 V_(H) SEQ ID NO: 89QIQLAQSGPELKKPGETVKISCKASGYTFTNYGMSWVKQAPGKGLKWMGWINTYTGEPTYAADFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARGYYDYWGQGTT LTVSS V_(L) SEQ ID NO: 90DVLMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGMFPRTFGGGTKLEIK

It is interesting to note that V_(L) CDR3 sequence is shared bySNP25₁₉₇-specific IgGs such as C4, C7, C14, C15, and C16 IgG, whereastheir V_(H) sequences tend to be IgG-specific (Table 11). Sequencealignment analysis revealed that not a single CDR sequence of IgGs,listed in Tables 10-12, overlaps with V_(L) and V_(H) CDR sequences ofpreviously reported IgGs (US patent U.S. Pat. No. 8,198,034B2). This maybe ascribed to employment of a more stringent screening strategy forpositive hybridoma cells in the present research. That is, throughoutmultiple rounds of screening, only triple positive hybridoma clones wereselected by direct ELISA using peptide antigen, sandwich ELISA with TCL,and Western blot analysis with TCL. This stringent screening strategymust have contributed to significantly lower K_(D) values of IgGsobtained in the present research (see below).

Example 11: Kinetics Analysis of Monoclonal Antibodies

As detailed in Materials and Methods, the kinetics analysis ofmonoclonal antibody was exclusively carried out by the BLI assay usingFORTÉBIO® Octet Red96 instrument, following the procedure recommended bythe manufacturer. Firstly, kinetics analysis was performed withSNAP25₁₉₇-specific IgGs using anti-GST biosensors loaded withrecombinant GST-SNAP25₁₉₇ (125 nM or 250 nM). FIG. 15 shows a set ofkinetics curves obtained with C4, C7, C16, and C24, as an example ofsuch studies. In brief, anti-GST biosensors, loaded with GST-SNAP25₁₉₇,were sequentially dipped in 1× kinetics buffer, serially diluted IgGsamples (7.8, 15.6, 31.2, 62.5, 125, 250, 500 nM) (analyte association),and 1× kinetics buffer (analyte dissociation). Subsequent to associationand dissociation of analyte for 10 min each, raw data of kinetics curveswere obtained (FIG. 15a ). Kinetics curves were then aligned bysubtracting baseline BLI signal (FIG. 15b ), from which K_(D) wasestimated using FORTÉBIO® Octet analysis software (Table 14).

TABLE 14 SPR Kinetic Parameters (SNAP25₁₉₇) Monoclonal Abs K_(on)(M⁻¹s⁻¹) K_(dis) (s⁻¹) K_(D) (nM) SNAP25₁₉₇ C4 2.30 × 10⁵ 4.10 × 10⁻³17.8 ± 4.45 specfic C7 2.57 × 10⁵ 4.94 × 10⁻³ 19.3 ± 3.15 (dipped in C162.35 × 10⁵ 2.15 × 10⁻³ 9.17 ± 2.71 125 nM) C24 5.29 × 10⁵ 5.09 × 10⁻³9.62 ± 1.30 SNAP25₁₉₇ C4 2.27 × 10⁵ 1.48 × 10⁻³ 6.53 ± 1.09 specific C72.77 × 10⁵ 1.22 × 10⁻³ 4.41 ± 0.68 (dipped in C16 2.36 × 10⁵ 1.52 × 10⁻³6.44 ± 1.25 250 nM) C24 4.58 × 10⁵ 2.41 × 10⁻³ 5.25 ± 1.09

Considering the nM range of K_(D) values, kinetics analysis seemed tonormally proceed. However, when kinetics analysis was performed with abiosensor dipped in a lower concentration of GST-SNAP25₁₉₇ (125 nM),K_(D) values proportionally decreased, mainly due to changes indissociation rate constant Kdis (Table 14). It was also at odd that Kdisvalues (1.22-5.09×10⁻³) were 1 or 2 orders of magnitude lower thanpreviously reported ones (3.11×10⁻⁴-6.74×10⁻⁵) for monoclonal antibodieswith a similar antigen specificity (US patent U.S. Pat. No.8,198,034B2).

Two plausible causes can be considered for relatively high K_(D) andKdis values. The first is the use of inappropriate assay buffer, and thesecond is the inherent limitation of the entire kinetics assay performedwith anti-GST biosensor (FortéBio Application Note 14: BiomolecularBinding Kinetics Assays on the Octet Platform). Comparative use ofdiverse assay buffers in the kinetics assay showed that 1× kineticsbuffer was the most appropriate among all buffers tested (data notshown). Therefore, as an alternative kinetics analysis, anti-mouse IgGFc capture (AMC) biosensors were directly loaded with antibody andsubjected to association/dissociation with serially diluted recombinantGST-SNAP25. In brief, purified IgG was diluted to 100 or 200 nM, whereasGST-SNAP25_(FL) or SNAP25₁₉₇ was serially diluted to 1.56, 3.125, 6.25,12.5, 25, 50, and 100 nM. After equilibration in 1× kinetics buffer for1 min, AMC biosensors were loaded with IgGs for 10 min, followed bydipping in 1× kinetics buffer for 10 min. Association and dissociationof analyte were carried out for 10 min each, and K_(D) was estimated asdescribed above.

Estimated K_(D) values using FORTEBIO® Octet analysis software arelisted in Table 15.

TABLE 15 Kinetics Parameters with SNAP25₁₉₇ Kinetics Parameters withSNAP25_(FL) IgGs K_(on) (M⁻¹ s⁻¹) K_(dis) (s⁻¹) K_(D) (pM) K_(on) (M⁻¹s⁻¹) K_(dis) (s⁻¹) K_(D) (pM) Bi-specific ^(a)A15 7.78 × 10⁴ 1.22 × 10⁻⁷1.56 ± 0.01 8.39 × 10⁴ 1.0 × 10⁻⁷ 1.19 ± 0.62 ^(a)B4  1.0 × 10⁵ 5.56 ×10⁻⁷ 0.48 ± 0.02 9.51 × 10⁴ 1.0 × 10⁻⁷ 0.10 ± 0.38 ^(a)B23 2.54 × 10⁴ 1.0 × 10⁻⁷ 3.27 ± 0.03 9.58 × 10⁴ 1.0 × 10⁻⁷ 0.10 ± 0.42 SNAP25₁₉₇-^(b)C4 9.28 × 10⁴  1.0 × 10⁻⁷ 1.07 ± 0.60 (not determined) specific^(b)C7 7.78 × 10⁴  1.0 × 10⁻⁷ 1.28 ± 0.75 ^(a)C14 7.67 × 10⁴  1.0 × 10⁻⁷1.30 ± 0.75 ^(b)C16 1.60 × 10⁶ 2.57 × 10⁻⁶ 1.62 ± 0.46 ^(a)C24 8.68 ×10⁴  1.0 × 10⁻⁷ 1.15 ± 0.87 ^(b)D3 1.11 × 10⁵  1.0 × 10⁻⁷ 0.90 ± 0.59SNAP25_(FL)- ^(b)D2 (not determined) 1.64 × 10⁵ 1.0 × 10⁻⁷ 0.61 ± 0.28specific ^(b)D6 3.52 × 10⁴ 1.0 × 10⁻⁷ 2.84 ± 0.02 ^(b)E6 5.38 × 10⁴ 1.0× 10⁻⁷ 1.86 ± 0.59

The most noticeable would be Kdis values (i.e. 2.57×10⁻⁶-1.0×10⁻⁷)(Table 15), which is two to three orders of magnitude lower than thoseobtained with anti-GST biosensor (Table 14). In fact, dissociation rateswere too low to be accurately measured using FORTÉBIO® Octet, so thelowest limit of Kdis value, 1.0×10⁻⁷, was tentatively given for sixIgGs, as described in Table 15.

Estimation of K_(D) values with these tentative dissociation rateconstants yielded 0.48-3.27 pM. It should be noted that AMC biosensorsloaded with SNAP25₁₉₇-specific IgGs did not show statisticallysignificant extent of association of GST-SNAP25_(FL) up to 1 μM.Similarly, GST-SNAP25₁₉₇ did not associate with AMC biosensors loadedwith SNAP25_(FL)-specific IgGs. These binding specificities of IgGs arein a good agreement with their reactivity in ELISA.

Example 12: Antigen Binding Specificity of Monoclonal Antibodies

Monoclonal antibodies were comparatively examined for their reactivitytoward SNAP25_(FL) and SNAP25₁₉₇ employing direct ELISA and Western blotanalysis. First, direct ELISA was performed with a purified IgG (50 ngper well) in a microplate coated with GST-SNAP25_(FL) or GST-SNAP25₁₉₇,as described in Materials and Methods. HRP reaction was carried out for5 min and the extent of HRP activity was determined by measuring A₄₅₀using Bio-Tek SynergyNeo2. A₄₅₀ values were obtained after subtractingbackground A₄₅₀, measured without IgG, and the ratio of A₄₅₀ withSNAP25₁₉₇ to A₄₅₀ with SNAP25_(FL) was calculated and presented asRatio_(197/206) in Table 16. Consistent with the result obtained by theBLI assay, Ratio_(197/206) values for bi-specific IgGs such as A15, B4,and B23, were close to 1.0. Ratio_(197/206) values for SNAP₁₉₇-specificIgGs such as C7, C14, C16, and C24, were over 950, butSNAP25_(FL)-specific IgGs yielded 0.02˜0.03 of Ratio_(197/206).

TABLE 16 A₄₅₀ with A₄₅₀ with IgGs SNAP25_(FL) SNAP25₁₉₇ Ratio_(197/206)A15 1.383 1.445 1.04 B4 2.163 1.797 0.83 B23 1.901 1.754 0.92 C4 0.0010.919 919 C7 0.001 1.199 1.037 C14 0.001 0.953 953 C16 0.001 0.996 996C24 0.001 0.973 973 D3 0.125 1.616 12.93 D2 1.448 0.048 0.03 D6 1.8780.031 0.02 E6 1.808 0.039 0.02

Western blot analysis was performed with hybridoma culture supernatants(1:100 dilution) as the source of primary antibody. Aliquots (0.5 μg) ofGST-SNAP25_(FL) and GST-SNAP25₁₉₇, resolved on a denaturing gel andsubsequently transferred to PVDF membrane, were tested as antigen.Monoclonal antibodies reacted with denatured SNAP25 antigen with thesame specificity as in the ELISA assay (FIG. 17). These results are notunexpected since only double positive hybridoma cells were selected by astringent ELISA and Western blot analysis.

Example 13: Conjugation of Horseradish Peroxidase (HRP) to Antibodies

Among monoclonal antibodies tested, C16 IgG exhibited the mostreproducible retention of antigenic affinity and specificity after beingconjugated with HRP (data not shown). In a typical HRP conjugationreaction, C16 IgG (5 mg) was incubated with activated HRP (5 mg) for 2hr at RT with light protection, as described in Materials and Methods.After the addition of 0.1 ml of sodium borohydride (4 mg/ml), theHRP-antibody conjugate was dialyzed against 1×PBS and 1×PBS/50% glycerolat 4° C. As shown in FIG. 18a , conjugation of activated HRP to C16 IgGwas very efficient that the formation of C16 IgG-HRP conjugate wasdetectable even without incubation (compare lanes 1-3). Incompleteconjugation of C16 IgG, reflected as free IgG and HRP (lanes 4-6),suggests the requirement of relatively high concentrations of free HRPand IgG in the reaction mixture for efficient conjugation.

Two different C16 IgG-HRP conjugates were examined for the reactivitytoward SNAP25₁₉₇ in direct ELISA. A 96-well microplate was coated with0-200 pg of GST-SNAP25₁₉₇, mimicking approximately up to ˜0.8% cleavageof endogenous SNAP25 in cells grown in a microplate well. Aliquots (200ng) of C16 IgG-HRP conjugates were capable of detecting as low as 50 pgof GST-SNAP25₁₉₇ when measured for 30 min with 50 μl of 1-Step™ UltraTMB-ELISA (FIG. 18b ). Also, higher A₄₅₀ values were obtained inproportion with increased amounts of GST-SNAP25₁₉₇. Based on theseresults, C16-HRP conjugates were exclusively used as detection antibodyin the optimized sandwich ELISA.

Example 14: Conjugation of Biotin to Antibodies

Sandwich ELISA developed by Allergan utilizes two antibodies: aSNAP25₁₉₇-specific IgG as capture antibody and polyclonalSNAP25-specific IgG as detection antibody. By contrast, sandwich ELISAinvented by the present research utilizes two monoclonal antibodies ascapture either detection antibody, which makes its quality control morefeasible and easier. This novel sandwich ELISA exhibits high levels ofrepeatability, reproducibility and accuracy. Yet the addition of asecond detection antibody such as IgG conjugated with alkalinephosphatase or biotin could further improve the accuracy of sandwichELISA through normalization of SNAP25 captured in each well.

As the first attempt, a bi-specific monoclonal antibody A15 wasconjugated with biotin, as described in Materials and Methods. As shownin FIG. 19 the higher biotin concentration was provided, the more biotinmolecules were conjugated per IgG. Quantitation of biotin conjugationusing HABA/Avidin Premix (Thermo Scientific) revealed that about 8 molesof biotin were conjugated per mole of IgG in a reaction provided with0.25 mM biotin. Consistently, the heavy chain of IgG conjugated with 0.5mM biotin exhibited noticeably slower migration on SDS-PAGE (FIGS. 19a &19 b).

The reactivity of A15 IgG conjugated with 0.1 mM biotin was tested insandwich ELISA. In brief, TCLs (60 μl) were prepared from N2-42F cellstreated with the indicated concentration of BoNT/A. A microplate coatedwith B4 IgG was incubated with 50 μl aliquots of TCL, and. endogenousSNAP25₁₉₇ captured by B4 IgG was detected by incubation with A15-biotinconjugate and streptavidin-AP conjugate (1:500 dilution in 1×TBS). As abi-specific monoclonal antibody, A15 IgG has been characterized to bindboth SNAP25_(FL) and SNAP25₁₉₇ with comparable affinity and specificity(see FIG. 17). These properties are among criteria to select A15 IgG asthe potential second detection antibody for the purpose ofnormalization. Thus, it was expected that A15 binding to SNAP25 remainrelatively unaffected by the extent of SNAP25 cleavage. On the contrary,however, AP activity, reflecting the SNAP25 binding of biotinylated A15IgG, increased in proportion to BoNT/A concentration (FIG. 19c ). Thisresult might be explained by the change in its antigen specificity uponbiotinylation of both light and heavy chains of IgG (FIG. 19b ).

Example 15: Direct Cross-Linking of Alkaline Phosphatase (AP) toAntibodies

As an alternative approach to the second detection antibody, purifiedIgGs were directly cross-linked to AP using glutaraldehyde as the extentof cross-linking can be regulated by glutaraldehyde concentration. Twopolyclonal and three monoclonal antibodies were cross-linked to AP usingglutaraldehyde. Except C16, they were all bi-specific antibodiesreacting with both SNAP25_(FL) and SNAP25₁₉₇, a key property as thesecond detection antibody for normalization. C16 IgG was used as controlsince HPR conjugation was efficient, yet not affecting the antigenspecificity of C16 (FIG. 18). In search for an optimal condition toobtain AP-IgG conjugate retaining its antigen specificity andreactivity, crosslinking of AP to IgG was performed with diverse ratiosof IgG to AP under different incubation conditions (Table 17).

TABLE 17 Ratio of Incubation Antibody IgG to AP time/temperatureAnalysis Cl 1:3 2 hr/4° C. + 2 hr/RT SDS-PAGE C16 1:1~2:1 3-24 hr/RT or37° C. A15 1:1~2:1 5 hr/37° C. SDS-PAGE rA15 2:1 2 hr/4° C. hr/RT &direct ELISA Sigma IgG 2:1 1-4 hr/4° C. or RT

Upon completion of glutaraldehyde cross-linking, the resulting AP-IgGconjugates were analyzed by SDS-PAGE. A representative result showingthe electrophoretic resolution of AP-IgG conjugates and the subsequentvisualization by Coomassie blue staining is shown in FIG. 20. In brief,polyclonal anti-SNAP25 IgG (Sigma) was cross-linked to AP in a reactionprovided with 0.2% glutaraldehyde. Cross-linking of AP was highlyefficient that all IgGs provided formed high molecular weight complexeswith AP. The resulting AP-IgG conjugates exhibited much slower migrationon a SDS gel, and some failed to enter the stacking gel portion (FIG.20a ). Similar patterns were obtained with all IgGs tested (data notshown).

Following the confirmation of the cross-linking by SDS-PAGE analysis,AP-rA15 IgG and AP-Sigma IgG conjugates (100 ng per well) werecomparatively examined for the antigen reactivity and specificity indirect ELISA using a microplate coated with 2 ng of GST-SNAP25 mixturescontaining varying extents of GST-SNAP25₁₉₇. AP-Sigma IgG conjugates didnot yield any ELISA signal, whereas AP-rA15 IgG conjugates led to aresult similar to that with biotinylated A15 IgG (compare FIG. 19 andFIG. 20b ). With increasing amounts of GST-SNAP25₁₉₇, higher ELISAsignals were obtained with AP-rA15 IgG conjugates.

The results in FIGS. 19 & 20 suggest that in case ofSNAP25_(FL)-reacting antibodies, be it monoclonal or polyclonal, bothheavy and light chains of IgG are efficiently conjugated to activatedbiotin or cross-linked to AP by glutaraldehyde. Thus, the seconddetection antibody remains to be developed until a more Fc-specificconjugation technology is available.

Example 16: Optimization of Culture of N2-42F, and Treatment with BoNT/A

Having acquired key reagents such as neuronal cells highly sensitive toBoNT/A and monoclonal antibodies specific for SNAP25, a series ofexperiments were carried out to optimize all steps in the cell-basedpotency assay, including the intoxication medium, sensitizer, BoNT/Atreatment time, and capture/detection antibody pairing.

Example 16-1. Optimization of BoNT/A Intoxication Time

First, the BoNT/A processing time (toxinization time) was optimized.

Protocol A (FIG. 21a ) is a standardized CBPA procedure optimized forSiMa. To examine the BoNT/A sensitivity following Protocol A, N2-42Fcells were plated at 5.6×10⁵ cells per well in a 12-well culture plate,and on next day, the medium was replaced with 1× intoxication mediumwithout GT/1b. Two days later, medium was supplemented with GT1b (25mg/ml), and after one more day of incubation, culture medium wasreplenished with 1× intoxication medium containing BoNT/A and incubatedfor additional 2 days. Protocol B (FIG. 21b ) is a CBPA proceduredeveloped in this research. In brief, N2-42F cells were plated at5.6×10⁵ cells per well in a 12-well culture plate, and on next day, themedium was replaced with 1× intoxication medium containing 25 pM BoNT/A.Total cell lysates were prepared on the indicated day by adding 1×SDSsample buffer (200 μl per well) and stored at −20° C. before use.Aliquots (12 μl) were subjected to 12% SDS-PAGE, and SNAP25_(FL) andSNAP25₁₉₇ were detected by Western blotting using polyclonal anti-SNAP25IgGs (Sigma 59684, 1:8,000 dilution) and goat anti-rabbit IgG Fc-HRP(AbFrontier LF-SA8002, 1:8,000 dilution). The extent of SNAP25 cleavagewas quantified using the Image Lab software (Bio-Rad).

As described above, towards establishment of the optimal intoxicationtime for N2-42F cells, Protocol A was modified by prolonging the cellculture time in either 1× intoxication medium devoid of GT1b or in 1×intoxication medium containing BoNT/A. These changes did not improve theextent of SNAP25 cleavage and moreover, N2-42F cells grown in the 1×intoxication medium for more than 4 days looked very unhealthy under themicroscope (data not shown). Based on this observation, a novel ProtocolB was established, where the culture time in 1× intoxication mediumsupplemented with both GT1b and BoNT/A was shortened (FIG. 21b ). Withthe lapse of day, starting from the third day (d4) subsequent to thecell plating, the extents of SNAP25 cleavage in N2-42F cells wascomparatively analyzed by Western blot. As shown in FIG. 21c , less than20% of SNAP25 cleavage was estimated on d4, but the prolonged culture ofN2-42F cells beyond d4 in 1× intoxication medium supplemented with GT1band BoNT/A led to significant increase in the SNAP25 cleavage up to 64%on d7. Since N2-42F cells on d7 looked unhealthy under the microscope,d6 was determined as the day of N2-42 cell harvesting and sandwich ELISAanalysis to measure the BoNT/A potency.

Example 16-2. Optimization of Culture Medium

The osmolarity and temperature influenced the BoNT/A sensitivity ofBOCELL™ (US patent U.S. Pat. No. 9,526,345B2). Also, the BoNT/Asensitivity of NG108-15 cells was significantly improved by optimizingneural differentiation medium (J Biomol Screen. 2016 January;21(1):65-73). Since the BoNT/A sensitivity reflects the extent of BoNT/Auptake through two independent receptors on cell surface, apolysialoganglioside (PSG) receptor and a protein receptor (SV2) (JNeurochem. 2009 June; 109(6):1584-95), the enhanced BoNT/A sensitivityby optimization of culture medium or higher temperature has to do withthe facilitated cellular intake of BoNT/A. To this end, N2-42F cells wastested for the BoNT/A sensitivity in three different culture media:RPMI1640, NEUROBASAL™, and MEM, all supplemented with 1×N2, 1×B27, and1×GT1b. While culturing in the indicated medium, N2-42F cells weretreated with varying concentrations (0.93˜25 pM) of BoNT/A cellsaccording to Protocol B. When measured by Western analysis, the BoNT/Asensitivity of N2-42F cells was measured the highest with RPMI1640 (FIG.22). The BoNT/A sensitivity measured with NEUROBASAL™ or MEM was 25% or50% lower than with RPMI1640. The KCl content is commonly 5.33 mM in allmedia, but the NaCl concentration is 103 mM in RPMI1640, 117 mM in MEM,and 52 mM in NEUROBASAL™ medium. Despite this difference, the osmolalityof cell culture media for most vertebrate cells is known to be keptwithin a narrow range from 260 mOsm/kg to 320 mOsm/kg (ATCC Culture CellGuide). Thus, the BoNT/A sensitivity of N2-24F cells in RPMI1640 islikely to be contributed by as yet unidentified medium component otherthan the osmolarity.

Under the condition described in FIG. 22, about 48% of endogenous SNAP25was cleaved in N2-42F cells by 8.33 pM BoNT/A, equivalent to about 10units/ml potency. Since the culture volume in a 96-well plate is 0.1 ml,the EC50 can be estimated to be ˜1 U bio-potency of BoNT/A per well in amicroplate. Thus, the BoNT/A sensitivity measured with N2-42F cellsfollowing Protocol B is sensitive enough to measure the bio-potency ofBoNT/A determined by the mouse LD50 bioassay.

Example 16-3. Identification and Optimization of Sensitizers

Taking advantage of having fully characterized monoclonal antibodiesspecific for SNAP25 (see below), the BoNT/A sensitivity of N2-42F cellsin the 1× intoxication medium containing varying concentrations ofBoNT/A (0.03-5.5 pM) were examined by sandwich ELISA assay followingProtocol B, while testing if the BoNT/A sensitivity is affected byarginine (see above) or any compounds with demonstrated effects onneural survival or differentiation, including ATP (Trends Neurosci. 2000December; 23(12):625-33), creatine (J Neurochem. 2005 October;95(1):33-45), and lipoic acid (J Neurosci Res. 2014 January;92(1):86-94). As shown in FIG. 23a , the addition of 1 mM creatine or 5mM arginine in the 1× intoxication medium noticeably enhanced the BoNT/Asensitivity, lowering EC50 value from 2.51 pM to 2.13 or 2.03 pM,respectively. By contrast, ATP and lipoic acid acted as very effectiveinhibitors that the SNAP25 cleavage in N2-42F cells was not detectedeven with 25 pM BoNT/A.

In an optimization study for the toxinized medium, it was believed thatthe BoNT/A sensitivity of N2-24F cells was affected by factors otherthan osmotic pressure. Because the sensitivity was higher in RPMI1640medium than in Neurobasal™ or MEM medium. For arginine in the mediumcomposition, it contained 1.15 mM in RPMI1640, 0.6 mM in MEM, and 0.4 mMin Neurobasal™. Since arginine is a precursor amino acid of creatine,arginine alone was further examined for its effects on the BoNT/Asensitivity using 2× intoxication medium containing 2-10 mM arginine. Asshown in FIG. 23b , the BoNT/A sensitivity was gradually enhanced withincreasing arginine concentration up to 5 mM, as reflected by loweredEC50 values. The BoNT/A sensitivity with 10 mM arginine (EC50=2.34 pM),though still higher than control (EC50=2.94 pM), was lower than with 5mM arginine (EC50=1.65 pM). Based on this result, although its mechanismof action remains yet to be understood, the optimized standard protocolof CBPA uses the intoxication medium containing 5 mM arginine.

In 2002, Schengrund and his coworkers provided experimental evidence forthe first time that in neuro-2a cells, an efficient SNAP25 cleavage byBoNT/A requires higher than 25 μg/ml GT1b in DMEM (J Biol Chem. 2002Sep. 6; 277(36):32815-9). Since N2-42F cells are derived from neuro-2a,the requirement of GT1b for BoNT/A activity was examined using the 1×intoxication medium containing 25-75 μg/ml GT1b (1˜3×GT1b) by Westernblot analysis. Without GT1b supplementation, 8.3 pM BoNT/A led to about18% of SNAP25 cleavage (FIG. 24a ). Addition of 1× or 3×GT1b resulted in10% and 18% increase in SNAP25 cleavage by 8.3 pM BoNT/A, respectively,in N2-42F cells (FIG. 24a ).

Towards optimization of GT1b concentration, the BoNT/A sensitivity ofN2-42F cells were tested in 2× intoxication medium containing 25 pMBoNT/A and increasing concentrations of GT1b from 1× to 5×. Whenmeasured by Western blot analysis, the SNAP25 cleavage was significantlyenhanced by the addition of 1× or 2×GT1b, but with more than 2×GT1b, theSNAP25 cleavage only marginally increased from 63%, 65%, 68%, to 70%(FIG. 24b ). A parallel test was performed employing the optimizedsandwich ELISA. Considering the sensitivity of sandwich ELISA, N2-42Fcells were treated with 0.93 pM BoNT/A in 2× intoxication mediumfollowing Protocol B. Sandwich ELISA more profoundly exhibited therequirement of GT1b for the BoNT/A activity. In brief, when N2-42F cellswere treated with BoNT/A in the intoxication medium lacking GT1b,relatively low A₄₅₀ values were obtained in sandwich ELISA (FIG. 24c ).By contrast, the addition of GT1b to the intoxication medium led tomarked increases in A450 value. The result showing a steady increase inA₄₅₀ value with up to 2×GT1b may reflect efficient and stabletri-molecular interaction between BoNT/A, GT1b, and polysialoganglioside(PSG) receptor. Therefore, saturated A₄₅₀ value obtained with 4×GT1b andeven decreased A₄₅₀ value with 5× may be explained by saturation of PSGreceptor and/or the effect of molar excess of free GT1b that competeswith BoNT/A-GT1b complex for PSG receptor. Based on this reasoning, 2×,that is 50 μg/ml, was selected as an optimal concentration of GT1b whenthe BoNT/A activity is measured using N2-42F cells.

The effect of N2(N2 supplement, Thermo Fisher Scientific17502048)/B27(B27™ Serum free supplement, Thermo Fisher Scientific17504-044) in neuron cultures on BoNT/A sensitivity was tested. B27,containing all trans-retinol (0.1 mg/L), is known to support motorneuron differentiation of neural progenitor cell (J Cell Biochem. 2008Oct. 15; 105(3):633-40). N2 contains a subset of B27 components,including insulin and is known to promote (1) differentiation of humanembryonic stem cells and (2) proliferation/survival of neural progenitorcell (J Cell Biochem. 2008 Oct. 15; 105(3):633-40). The BoNT/Asensitivity of N2-42F cells in RPMI1640 supplemented with 1×GT1b, 8.3 pMBoNT/A and the indicated concentration of N2/B27 following Protocol B.The SNAP25 cleavage was quantitatively analyzed by Western blot. Asshown in FIG. 25a , intracellular levels of total SNP25(SNAP25_(FL)+SNAP25₁₉₇) increased in proportion to N2/B27 concentration,whereas the level of SNAP25197 remained relatively unchanged and evendecreased in the presence of 4× or 5×N2/B27. A recent study reportedthat N2 and B27 function jointly to protect neuron from cell death afterglucose depletion by restricting glycolysis (Front Mol Neurosci. 2017Sep. 29; 10:305). Consistently, the result in FIG. 25a is very likelyreflect the enhanced synthesis of endogenous SNAP25 with increasingconcentrations of N2/B27 as part of their function to promote cellproliferation and survivor in serum-free RPMI1640 medium containingrelatively low concentration of glucose (11.1 mM).

Using RPMI1640 supplemented with 3×GT1b and 5 mM arginine, the effectsof N2/B27 on the BoNT/A activity were further examined. The extent ofSNAP25 cleavage in N2-42F cells treated with 8.3 pM BoNT/A was enhancedfrom 13% to 56%, 69% by 1× and 2×N2/B27 in spite of noticeable increasein overall intracellular level of SNAP25 (FIG. 25b ). Based on thisobservation, 2×N2/B27 is added to the optimized intoxication medium.

Example 17: Optimization of Sandwich ELISA Example 17-1. Optimization ofBuffers

Having optimized the culture medium and media supplements for N2-42Fcell intoxication, next, sandwich ELISA was systematically evaluated forits performance under diverse conditions summarized in Table 18. Andselected optimized conditions summarized in Table 19.

TABLE 18 Parameters Conditions tested Optimal condition Plate coating 3matrices Poly-D-lysine matrix Cell density 2 densities 5.5 × 10⁴ cellsper well Intoxication 3 culture media/ RPMI1640, 2 mM medium/ 6sensitizers L-alanyl-L-glutamine, sensitizer 0.1 mM NEAA, 2x N2, 2x B27,GT1b (50 μg/

,) 5 mM arginine Intoxication time 3 time points 96 hr BoNT/A dose 0.03pM-1 nM 0.03-8.33 pM Capture/ 11 combinations B4 (300 ng/50

) + C16-HRP detection antibodies (200 ng/50

) Lysis buffers 11 buffers 20 mM Hepes-NaOH, pH 7.5, 0.2 M NaCl, 1%TRITON X-100, 1 mM EGTA, 5 mM EDTA Lysate incubation 3 temperatures/ 4hr at 4° C. 3 time points Blocking buffers 46 buffers 1% polyvinylalcohol (Mw 145,000), 3% skim milk in 1x PBS Incubation of 4temperatures/ 1 hr at RT detection antibody 4 time points

TABLE 19 Parameters Condition tested Optimal Lysis buffers 50 mMTris-HCl, pH 8.0,150 mM NaC1, 1% TRITON 20 mM Hepes- X-100, 2 mM EGTA,0.01-0.1% SDS, ± 5 mM EDTA NaOH, pH 7.5, 20 or 50 mM Hepes-NaOH, pH 7.5,0-0.4 M NaCl, 1 or 150 mM NaCl, 2% TRITON X-100, 0-0.1% SDS, 1% TRITONX- 0-1.5 mM MgCl₂, 0-5 mM EDTA, 1-2 mM EGTA 100, 5 mM EDTA, 50 mMTris-HCl, pH 8.0, 150 mMNaCl, 0.1% SDS, 2 mM EGTA 0.5% sodiumdeoxycholate, 1% NP-40 Coating buffers 0.5% APTES 0.1 M carbonate 0.1 Msodium acetate buffer, pH 6.0 buffer, pH 9.6 0.1 M sodium phosphatebuffer, pH 7.2 0.1 M sodium citrate buffer, pH 2.8 0.1 M carbonatebuffer, pH 9.6 Washing buffers 1x PBS/0.05% TWEEN-20 (1x PBST), ± 0.2 MNaCl 1x PBST Blocking 0.2-3% Ac-BSA, 3-5% skim in 1x PBS 3% skim,buffers 1% goat serum in 1x PBS 1% PVA 2-3% skim, 0.05-1% PVA (Mw 47,000or 145,000) (Mw 145,000) in 1x PBS in 1x PBS 5% skim or 0.1-0.5% Ac-BSA,with 10 or 100% SUPERBLOCK ™ (PBS)^(a) 2-5% ECL^(b) or ECL PRIME ™Blocking agent^(c) ± 0-5% skim or 0.2% Ac-BSA Western BLoT blockingbuffer (protein-free)^(d) ^(a) SUPERBLOCK ™ (PBS) (Thermo FisherScientific 37518) ^(b) ECL PRIME ™ blocking agent (GE Healthcare UKlimited RPN418V) ^(c) ECL (GE Healthcare UK limited RPN2125V) ^(d)Western BLoT blocking buffer (protein free) (Takara T7132A)

Example 17-2. Optimization of Capture Antibody

Secondly, three bi-specific antibodies, A15, B4, and B23 were comparedfor their function as capture antibody in sandwich ELISA. TCLs wereprepared from N2-42F cells treated with varying concentrations (0-25 pM)of BoNT/A in 1× intoxication medium. TCLs were added to a microplatecoated with the indicated antibody (400 ng per well). After incubationfor 4 hr at 4° C., the amount of SNAP25₁₉₇ among total SNAP25 capturedby the indicated antibody was detected and quantified using C16 IgG-HRPconjugates following the procedure described in Materials and Methods.One exception was that the HRP reaction was carried out for 30 min untilall test groups yielded positive ELISA signal. As shown FIG. 26a , theestimated EC50 values were 15 pM, 0.7 pM, and 5.5 pM for A15, B4 and B23IgG, respectively. This result is consistent with their K_(D) values forSNAP25_(FL) and SNAP25₁₉₇.

Optimal B4 IgG quantity was explored with TCLs prepared from N2-42Fcells incubated in 2× intoxication medium containing the indicatedconcentration of BoNT/A following Protocol B. Sandwich ELISA wasperformed with a microplate coated with increasing amounts (100˜400 ngper well) of B4 IgG. After incubation for 4 hr at 4° C., SNAP25₁₉₇captured was detected and quantified using C16 IgG-HRP conjugates asdescribed above. It should be noted that the HRP reaction was carriedout for 15 min. As shown FIG. 26b , EC50 value was steadily lowered withincreasing B4 IgG quantity from 100 ng to 300 ng. There was no furtherstatistically significant improvement in EC50 with 400 ng of B4 IgG.Taken together, 300 ng of B4 IgG is used as the standard quantity ofcapture antibody in the optimized sandwich ELISA.

Example 17-3. Optimization of Incubation Conditions for TCL andDetection Antibody

TCLs, prepared from SiMa cells treated with 8.33 or 25 pM BoNT/A in 1×intoxication medium, were incubated in a microplate under the indicatedcondition. Subsequently, SNAP25₁₉₇ captured was detected and quantifiedusing C16 IgG-HRP conjugates. As shown in FIG. 27a , ELISA signal forSNAP25₁₉₇ was the highest when the microplate was incubated for 4 hr at4° C. but was very weak after incubation at 37° C. for 1 hr. Althoughdata not shown, similar results were obtained with TCLs of N2-42F cells,and ELISA signal was not significantly changed by longer than 4hr-incubation. Thus, in the optimized sandwich ELISA, TCL is incubatedfor 4 hr at 4° C.

The optimal incubation condition for detection antibody was explored bydirect ELISA. In brief, the microplates were coated with varying amounts(from 10 pg to 1 ng) of recombinant GST-SNAP25₁₉₇ that represent theendogenous SNAP25 cleavage from 0.05% to 50% in the standard CBPA. Afteradding C16 IgG-HRP conjugates (200 ng per well), the microplates wereincubated under the indicated condition. Of conditions test, incubationof Cg16 IgG-HRP conjugates at RT for 1 hr yielded the highest ELISAsignal for all ranges of GST-SNAP25₁₉₇ examined but other conditionsalso generated acceptable levels of ELISA signal. When considering thesubsequent HRP reaction that is carried out at RT, however, in theoptimized sandwich ELISA, C16-HRP conjugates are incubated for 1 hr atRT.

Example 17-4. Optimization of Detection Method for C16 IgG-HRPConjugates

HRP activity was generally measured using a conventional TMB substrate(1-STEP™ Ultra TMB-ELISA). One drawback in using TMB substrate is EC50value tends to be influenced by HRP reaction time (FIGS. 26 & 29). Thelonger HRP reaction yields the lower EC50. As an alternative detectionmethod, a fluorimetric measurement was comparatively evaluated insandwich ELISA. Except for the HRP reaction substrate provided, thecolorimetric and fluorimetric measurement were carried out in parallelfollowing essentially the same way throughout sandwich ELISA.Fluorimetric and colorimetric measurements generated 0.66 and 0.78 pM ofEC50 (FIG. 28), and both EC50 values were equally influence by HRPreaction time (data not shown). In conclusion, despite the fluorimetricmeasurement of HRP reaction necessitates a black microplate and amicroplate reader built-in with a fluorimeter, both measurements areaccurate and sensitive enough to detect the activity of C16 IgG-HRPconjugates in the optimized ELISA.

Example 18: Validation and Practical Application of CBPA Example 18-1.Optimized Timeline of CBPA

Taken all optimized conditions and reagents together, the finallyestablished optimal CBPA can be performed with only three working days,as depicted in FIG. 29. In brief, cells are plated on day 1, and on thenext day 2, medium is replaced with 2× intoxication medium containingBoNT/A (0.1-10 U/ml or less than 4 pM). On day 6, cells are treated withlysis buffer, and with the resulting TCL, sandwich ELISA is carried outand BoNT/A bio-potency is determined. Avoiding the bench-work onweekend, it is possible to perform the optimized CBPA three times aweek, with only 1-2 working hours during the first two days and onefull-working day. Thus, the optimized CBPA is suitable for measuring thebio-potency of multiple lots of pharmaceutical or cosmetic BoNT/Aproducts a week. The use of two monoclonal antibodies for sandwichELISA, excluding rabbit polyclonal antibodies, makes the CBPA highlyreliable since their acquisition and subsequent quality control aresuperior to those of polyclonal antibodies.

Example 18-2. Accuracy and Linearity of CBPA

Following the standard Protocol B, a total of 18 CBPA were performed bythree operators.

A total of 18 CBPA were performed by three operators following ProtocolB. N2-42F cells were treated with varying concentrations (0, 0.03, 0.1,0.2, 0.31, 0.62, 0.93, 2.78, 5.55, 8.33 pM) of BoNT/A in 2× intoxicationmedium (FIG. 30a ) or 2× intoxication medium containing 3×GT1b (FIG. 30b& FIG. 30c ). HRP reaction was carried out for 5 min (FIG. 30a & FIG.30b ) or 9 min (FIG. 30c ), and EC50 was determined using Gen5 software.Linear regression analysis was performed with A₄₅₀ values obtained withN2-42F cells treated with 0.1 to 0.93 pM BoNT/A, and the results areshown in FIG. 30.

The first series of CBPA yielded 1.39 pM of EC50 with 1.5 fM (3.4 mUrelative potency/ml) of detection limit (DL) and 4.6 fM of quantitationlimit (QL) (Clin Biochem Rev. 2008 August; 29 Suppl 1:S49-52; Anal Sci.2007 February; 23(2):215-8). Since the 1.39 pM BoNT/A is equivalent to˜0.3 U relative potency per assay, the first CBPA is sensitive enough tomeasure the bio-potency of BoNT/A in place of mouse LD50 assay. Aslightly lower EC50 value, 1.24 pM, was obtained by a prolonged HRPreaction employed in the second series of CBPA (FIG. 30b ). The impactof HRP reaction time on EC50 was previously noticed (FIG. 29). The thirdseries of CBPA, performed using the intoxication medium with 3×GT1b,yielded the lowest 1.09 pM of EC50, consistent with the results in FIG.27.

This result indicates that the quantitation power and detection limit ofCBPA, developed in the present research, can be adjusted by modulatingHRP reaction time or changing GT1b concentration in the intoxicationmedium. Linear regression analysis of ELISA signals for N2-42F cellstreated with 0.03˜0.93 pM BoNT/A revealed an excellent linearcorrelation between the relative potency of BoNT/A and ELISA signal inall three series of CBPA (FIG. 30). This result indicates that CBPA isnot only highly sensitive but also very accurate in measuring therelative potency of BoNT/A between 6.8 mU and 0.2 U per assay.

Example 18-3. Measurement of the Bio-Potency in BOTULAX® Samples

BoNT/A potency in BOTULAX® was determined by mouse LD50. To measure thisbio-potency using CBPA, two different lots (HUB 18009 and HUB 18011) ofBOTULAX® (200 U per vial) were dissolved in either intoxication medium(matrix a), deionized H₂O (matrix b), or 5 mM arginine, pH 6.0 (matrixc). After 10 min incubation at RT, BOTULAX® dissolved in medium wasserially diluted to 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, and 50 U/ml, butthe others were to 0.23, 0.48, 0.70, 0.98, 1.41, 2.11, 4.20, 6.32, and12.6 U/ml in the optimized intoxication medium. Three different sets ofCBPA were carried out using these samples following the standardProtocol B. The EC50 values was determined. As shown in FIG. 31, EC50values of two lots were 10.6±0.12 and 10.3±0.52 U/ml with matrix a,4.6±0.04 and 4.6±0.08 U/ml with matrix b, and 4.5±0.11 and 4.4±0.22 U/mlwith matrix c. This result indicates that CBPA is sensitive and accurateenough to measure the bio-potency of BOTULAX® and its sensitivity(0.4-0.5 U per well) is equivalent or superior to the mouse bioassay.

Although the present disclosure has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only of a preferred embodimentthereof, and does not limit the scope of the present invention. Thus,the substantial scope of the present invention will be defined by theappended claims and equivalents thereof.

Accession Number

-   Depository authority: Korea Research Institute of Bioscience and    Biotechnology;-   Accession number: KCTC13712BP;-   Deposit date: Nov. 13, 2018.

N2-42F cell line (accession number: KCTC 13712BP) was deposited withKorea Research Institute of Bioscience and Biotechnology, on Nov. 13,2018. The subject cell line has been deposited under conditions thatassure that access to the cell line will be available during thependency of this patent application to one determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37CFR 1.14 and 35 U.S.C. 122. The deposit will be 10 available as requiredby foreign patent laws in countries wherein counterparts of the subjectapplication, or its progeny, are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

Further, the subject deposits will be stored and made available to thepublic in accord with 15 the provisions of the Budapest Treaty for theDeposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least thirty (30)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit 20 should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

1. A method for determining activity of botulinum toxin in a sample,comprising the steps of: (a) contacting a cell with the sample, whereinthe cell is from a cell line clonally derived from parental neuro-2acell (accession number KCTC AC28106), wherein the clonally derived cellline has homogenous cell population, and wherein the clonal cell linecomprises cells susceptible to intoxication by botulinum toxin type A(BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows highersensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to theparental neuro-2a cell under same condition, equal sensitivity to BoNT/Dof 5 pM or 200 pM concentration to the parental neuro-2a cell under samecondition, and no sensitivity to BoNT/E of 10-400 pM concentration; (b)obtaining cell lysate of the cell of (a), said cell lysate comprisingproteins of the cell of (a) or isolating proteins from the cell of (a);(c) contacting the cell lysate or the isolated proteins with an agentwhich specifically binds synaptosomal nerve-associated protein 25(SNAP25_(FL)) or botulinum toxin-cleaved SNAP25 fragment (SNAP25₁₉₇);(d) detecting the presence of a complex between the agent and theSNAP25_(FL) and/or SNAP25₁₉₇, and (e) determining the activity ofbotulinum toxin in the sample, wherein the higher the amount of theagent-antigen SNAP25_(FL) and/or SNAP25₁₉₇ complex detected the higherthe level of botulinum toxin activity in the sample.
 2. The method ofclaim 1, wherein the botulinum toxin is botulinum toxin type A (BoNT/A).3. The method of claim 1, which further comprises, prior to step (a),culturing the cell in a culture medium supplemented with gangliosideGT1b trisodium salt (GT1b).
 4. The method of claim 3, wherein theculture medium further comprises creatine and arginine.
 5. The method ofclaim 3, wherein the concentration of GT1b is 25-75 μg/ml.
 6. The methodof claim 4, wherein concentration of arginine is about 5 mM.
 7. Themethod of claim 1, wherein the agent is an antibody comprises: aheavy-chain CDR1 region which is any one selected from the groupconsisting of SEQ ID NOs: 11 to 13, 28 to 33, and 55 to 56; aheavy-chain CDR2 region which is any one selected from the groupconsisting of SEQ ID NOs: 14 to 16, 34 to 39, and 57 to 58; aheavy-chain CDR3 region which is any one selected from the groupconsisting of SEQ ID NOs. 17 to 19, 40 to 46, and 59 to 60; alight-chain CDR1 region which is any one selected from the groupconsisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62; alight-chain CDR2 region which is any one selected from the groupconsisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and alight-chain CDR3 region which is any one selected from the groupconsisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to
 66. 8. A methodfor detecting botulinum toxin in a sample, comprising the steps of: (a)contacting a cell with the sample comprising botulinum toxin orsuspected of comprising botulinum toxin, wherein the cell is from a cellline clonally derived from parental neuro-2a cell (accession number KCTCAC28106), wherein the clonally derived cell line has homogenous cellpopulation, and wherein the clonal cell line comprises cells susceptibleto intoxication by botulinum toxin type A (BoNT/A) by about 25 pM orless of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B,BoNT/C, and BoNT/F compared to the parental neuro-2a cell under samecondition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentrationto the parental neuro-2a cell under same condition, and no sensitivityto BoNT/E of 10-400 pM concentration; (b) obtaining cell lysate of thecell of (a), said cell lysate comprising proteins of the cell of (a), orisolating proteins from the cell of (a); (c) contacting the cell lysateor the isolated proteins with an agent which specifically bindssynaptosomal nerve-associated protein 25 (SNAP25_(FL)) or botulinumtoxin-cleaved SNAP25 fragment (SNAP25₁₉₇); (d) detecting the presence ofa complex between the agent and the SNAP25_(FL) and/or SNAP25₁₉₇; and(e) determining that when agent-antigen SNAP25_(FL) and/or SNAP25₁₉₇complex is detected, the botulinum toxin is present in the sample. 9.The method of claim 8, wherein the botulinum toxin is botulinum toxintype A (BoNT/A).
 10. The method of claim 8, which further comprises,prior to step (a), culturing the cell in a culture medium supplementedwith ganglioside GT1b trisodium salt (GT1b).
 11. The method of claim 10,wherein the culture medium further comprises creatine and arginine. 12.The method of claim 10, wherein the concentration of GT1b is 25-75μg/ml.
 13. The method of claim 11, wherein concentration of arginine isabout 5 mM.
 14. The method of claim 8, wherein the agent is an antibodycomprises: a heavy-chain CDR1 region which is any one selected from thegroup consisting of SEQ ID NOs: 11 to 13, 28 to 33, and 55 to 56; aheavy-chain CDR2 region which is any one selected from the groupconsisting of SEQ ID NOs: 14 to 16, 34 to 39, and 57 to 58; aheavy-chain CDR3 region which is any one selected from the groupconsisting of SEQ ID NOs. 17 to 19, 40 to 46, and 59 to 60; alight-chain CDR1 region which is any one selected from the groupconsisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62; alight-chain CDR2 region which is any one selected from the groupconsisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and alight-chain CDR3 region which is any one selected from the groupconsisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to 66.